EA033842B1 - Axmi477, Axmi482, Axmi486 AND Axmi525 TOXIN GENES AND METHODS FOR THEIR USE - Google Patents

Abstract

Compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds are provided. Compositions comprising a coding sequence for a toxin polypeptide are provided. The coding sequences can be used in DNA constructs or expression cassettes for transformation and expression in plants and bacteria. Compositions also comprise transformed bacteria, plants, plant cells, tissues, and seeds. In particular, isolated toxin nucleic acid molecules are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed, and antibodies specifically binding to those amino acid sequences. In particular, the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequence shown in SEQ ID NO: 5-26, or the nucleotide sequence set forth in SEQ ID NO: 1-4, as well as variants and fragments thereof.

Description

Link to related applications

This application claims the priority of US Provisional Application Serial Number 61/913905 filed December 9, 2013 and US Provisional Application Serial Number 61/913911 filed December 9, 2013, the entire contents of which are incorporated herein by reference .

Link to sequence listing filed electronically

The official copy of the list of sequences was submitted electronically via EFS-Web as a list of sequences in ASCII format, and a file of 97 kilobytes in size was registered on November 14, 2014 under the name APA136054_ST25.txt simultaneously with the specification. The sequence listing in this ASCII document is part of the specification and is incorporated by reference in its entirety.

Field of Invention

The present invention relates to the field of molecular biology. This document provides new genes that encode pesticidal proteins. These proteins and their nucleotide sequences encoding them are used for the preparation of pesticidal formulations and for the production of transgenic plants resistant to agricultural pests.

BACKGROUND OF THE INVENTION

Bacillus thuringiensis is a gram-positive spore-forming soil bacterium that has the ability to produce crystalline inclusions that exhibit specific toxicity with respect to certain orders and species of insects, but are harmless to plants and other non-target organisms. In this regard, compositions containing strains of Bacillus thuringiensis or their insecticidal proteins can be used as environmentally acceptable insecticides for the control of agricultural insect pests or vector insects for the treatment of various human and animal diseases.

Crystalline (Cry) proteins (delta-endotoxins) from Bacillus thuringiensis have potent insecticidal activity against insect larvae, which are predominantly part of the Lepidoptera and Semi-winged groups. Diptera and Coleoptera. These proteins also showed activity with respect to pest orders Hymenoptera, Homoptera, Phthiraptera, Mallophaga and Acari, as well as other invertebrate orders such as Nemathelminthes, Platyhelminthes and Sarcomastigorphora (Feitelson (1993) The Bacillus Thuringiensisered family tree Inners Advanced Parker. Inc., New York, NY) These proteins were initially classified as CryI-CryV, mainly based on their insecticidal activity. The main classes were Lepidoptera-specific (I), Lepidoptera-specific and Diptera-specific (II), Coleoptera-specific (III), Diptera-specific (IV) and nematode-specific (V) and (VI). Proteins are further classified into subfamilies; more closely related proteins in each family were assigned the letters Cry1A, Cry1B, Cry1C, etc., belonging to the subgroup. Even more closely related proteins within each subgroup received such designations as CryC1, Cry1C2, etc.

The nomenclature for Cry genes has been described based on homology of amino acid sequences rather than on target specificity for the insect (Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62: 807-813). According to this classification, each toxin is assigned a unique name, including the primary category (Arabic numeral), the secondary category (capital letter), the tertiary category (lowercase letter) and the Quaternary category (second Arabic numeral). Roman numerals have been replaced by Arabic numerals in the primary category. Proteins with sequence identity less than 45% have different primary categories, and the criteria for the secondary and tertiary categories are 78 and 95%, respectively.

Crystalline protein does not exhibit insecticidal activity until it is absorbed by the insect and dissolved in its middle intestine. The absorbed protoxin is hydrolyzed by proteases in the digestive tract of insects to form active toxic molecules. (Hofte and Whiteley (1989) Microbiol. Rev. 53: 242-255). This toxin binds to the apical receptors of the brush border in the middle intestine of the target larvae and integrates into the apical membrane, creating ion channels or pores, which as a result leads to the death of the larva.

Delta endotoxins generally have five conserved sequence domains and three conserved structural domains (see, for example, de Maagd et al. (2001) Trends Genetics 17: 193-199). The first conservative structural domain consists of seven alpha helices and is involved in penetration through the membrane and pore formation. Domain II consists of three folded beta layers that are ordered in the Greek key structure, and domain III consists of two anti-parallel folded beta layers in a jelly-roll type structure (de Maagd et al., 2001, see above). Domains II and III are involved in the recognition and binding of receptors and are therefore considered to be deviators of toxin specificity.

Due to the damage that insects can cause and to improve crop yields by controlling insect pests, there is a constant need to discover new forms of pesticidal toxins.

SUMMARY OF THE INVENTION

Provides compositions and methods for imparting pesticidal activity to bacteria, plants,

- 1,033,842 cells, tissues and seeds of plants. Compositions include nucleic acid molecules encoding sequences of pesticidal and insecticidal polypeptides, vectors containing these nucleic acid molecules, and host cells containing such vectors. The compositions also contain sequences of pesticidal polypeptides and antibodies to these polypeptides. Nucleotide sequences can be used in DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and plants. Nucleotide or amino acid sequences may be synthetic sequences that have been engineered for expression in the body, including, but not limited to, a microorganism or plant. Compositions also include bacteria, plants, cells, tissues and plant seeds containing the nucleotide sequence of the present invention.

In particular, isolated or recombinant nucleic acid molecules that encode a pesticidal protein are provided. In addition, amino acid sequences corresponding to the pesticidal protein are encompassed. In particular, the present invention provides an isolated or recombinant nucleic acid molecule that contains a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 5-26, or the nucleotide sequence set forth in SEQ ID NO: 1-4, as well as their biologically -active options and fragments. Nucleotide sequences that are complementary to the nucleotide sequence of the present invention, or which hybridize to the sequence of the present invention or its complementary strand, are also encompassed. Additionally provided are vectors, cell hosts, plants and seeds containing the nucleotide sequences of the present invention, or nucleotide sequences encoding the amino acid sequences of the present invention, as well as their biologically active variants and fragments.

Provides methods for producing the polypeptides of the present invention and the use of these polypeptides for the control or extermination of insects belonging to the orders Lepidoptera, Hemiptera, Coleoptera, nematodes or dipterans. Also included are methods and kits for detecting nucleic acids and polypeptides of the present invention in a sample.

The compositions and methods of the present invention are suitable for the production of organisms with increased resistance or resistance to pests. These organisms and compositions containing organisms are suitable for agricultural purposes. The compositions of the present invention are also suitable for creating altered or improved proteins that have pesticidal activity, or for detecting the presence of pesticidal proteins or nucleic acids in products or organisms.

Detailed description

The invention relates to compositions and methods for controlling resistance or resistance to pests in organisms, in particular, plants or plant cells. By resistance is meant that a pest (e.g., an insect) dies after ingestion or other contact with the polypeptides of the present invention. Tolerance means a violation or inhibition of the functions of movement, nutrition, reproduction or other functions of the body of the pest. The methods include transforming organisms using the nucleotide sequence encoding the pesticidal protein of the present invention. In particular, the nucleotide sequences of the present invention are used to obtain plants and microorganisms that have pesticidal activity. Thus, transformed bacteria, plants, plant cells, tissues and seeds of plants are provided. The compositions are pesticidal nucleic acids and proteins from Bacillus or other species. The sequences are used in the construction of expression vectors for subsequent transformation into organisms of interest as probes for the isolation of other homologous (or partially homologous) genes and for the creation of altered pesticidal proteins using methods known in the art, such as permutation of domain blocks or rearrangement in DNA, for example, using representatives of the endotoxin families Cry1, Cry2 and Cry9. Proteins are used in controlling or inducing the death of pest populations from orders of Lepidoptera, Semi-Rugged, Coleoptera, Diptera, and Nematodes, and for preparing compositions with pesticidal activity.

By the term pesticidal toxin or pesticidal protein is meant a toxin that has toxic activity with respect to one or more pests, including, but not limited to, members of the orders Lepidoptera, Diptera, Hemiptera and Coleoptera or of the type Nematoda, or a protein that is homologous to such a protein. Pesticidal proteins have been isolated from organisms that include, for example, Bacillus sp., Clostridium bifermentans and Paenibacillus popilliae. Pesticidal proteins contain amino acid sequences deduced from the full length nucleotide sequences disclosed herein and amino acid sequences that are shorter than the full length sequences either by using an alternative lower translation start site or by processing that results in a shorter protein characterized by pesticidal activity. Processing can take place in the body in which the protein is expressed, or in the pest after absorption of the protein.

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Thus, this document provides new isolated or recombinant nucleotide sequences that provide pesticidal activity. These nucleotide sequences encode polypeptides with homology to known delta endotoxins or binary toxins. Also provided herein are the amino acid sequences of pesticidal proteins. The protein synthesized as a result of the translation of this gene allows cells to control or cause the death of pests when they enter their digestive tract.

Isolated nucleic acid molecules and their variants and fragments

One aspect of the present invention relates to isolated or recombinant nucleic acid molecules that contain nucleotide sequences encoding pesticidal proteins and polypeptides or their biologically active parts, as well as nucleic acid molecules suitable for use as hybridization probes to identify nucleic acid molecules that encode proteins with regions of sequence homology. Also provided herein are nucleotide sequences capable of hybridizing to the nucleotide sequences of the present invention under stringent conditions, as defined elsewhere in this document. As used herein, the expression nucleic acid molecule includes DNA molecules (e.g., recombinant DNA, cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and DNA or RNA analogues created using nucleotide analogs. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.

The expression isolated or recombinant nucleotide sequence (or DNA) is used herein to refer to a nucleotide sequence (or DNA) that is no longer in its natural environment, for example, in an in vitro system or in a recombinant bacterial or plant host cell. In some embodiments, the isolated or recombinant nucleic acid does not contain sequences (preferably protein coding sequences) that naturally flank the nucleic acid (i.e., sequences that are located at the 5 'and 3' ends of the nucleic acid) in the genomic DNA the organism from which the nucleic acid is derived. In the context of this invention, the expression isolated, when used with respect to nucleic acid molecules, excludes the selected chromosomes. For example, in various embodiments, an isolated deltaendotoxin encoding a nucleic acid molecule may contain nucleotide sequences of less than about 5 thousand base pairs (kbp), 4 kbp, 3 kb ., 2 kbp, 1 kbp, 0.5 kbp, or 0.1 kbp, which under natural conditions flank the nucleic acid molecule in the genomic DNA the cell from which the nucleic acid was obtained. In various embodiments, a delta endotoxin protein that is substantially free of cellular material includes protein formulations containing less than about 30, 20, 10%, or 5% (dry weight) of the delta endotoxin protein (also referred to as contaminating protein).

The nucleotide sequences encoding the proteins of the present invention include the sequence set forth in SEQ ID NO: 1-4, and its variants, fragments and complementary chains. By complementary is meant a nucleotide sequence that is sufficiently complementary to a given nucleotide sequence so that it can hybridize with a given nucleotide sequence to form a stable duplex. The corresponding amino acid sequences of pesticidal proteins that are encoded by these nucleotide sequences are set forth in SEQ ID NO: 5-26.

Nucleic acid molecules, which are fragments of these nucleotide sequences encoding pesticidal proteins, are also included in the present invention. By fragment is meant a portion of the nucleotide sequence encoding a pesticidal protein. A fragment of the nucleotide sequence can encode the biologically active part of the pesticidal protein, or it can be a fragment that can be used as a hybridization probe or PCR primer using the methods described below. Nucleic acid molecules, which are fragments of a nucleotide sequence encoding a pesticidal protein, contain at least about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1350, 1400 contiguous nucleotides, or the number of nucleotides, reaching the number of nucleotides present in the full length nucleotide sequence encoding the pesticidal protein disclosed herein, depending on the intended use. By adjacent nucleotides is meant nucleotide residues that are directly adjacent to each other. Fragments of the nucleotide sequences of the present invention will encode fragments of proteins that retain the biological activity of the pesticidal protein and, therefore, retain pesticidal activity. Thus, the biologically active fragments of the polypeptides disclosed herein are also included in the present invention. By the expression “retains activity” is meant that the fragment will possess at least about 30%, at least about 50%, at least about 70, 80, 90, 95, or more pesticidal activity of the pesticidal protein. In one embodiment, the pesticidal activity is

- 3 033842 activity relative to beetles. In another embodiment, the pesticidal activity is relatively Lepidoptera activity. In another embodiment, the pesticidal activity is activity against nematodes. In another embodiment, the pesticidal activity is relatively diptera activity. In another embodiment, the pesticidal activity is relatively half-winged activity. Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83: 2480-2485; Andrews et al. (1988) Biochem. J. 252: 199-206; Marrone et al. (1985) J. of Economic Entomology 78: 290-293; and US patent No. 5743477, all of which are incorporated herein by reference in their entirety.

A fragment of the nucleotide sequence encoding the pesticidal protein that encodes the biologically active portion of the protein of the present invention will encode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 adjacent amino acids or up to the total number of amino acids present in the full-length protein of the pesticidal protein of the present invention. In some embodiments, the fragment is a fragment resulting from proteolytic cleavage. For example, a fragment resulting from proteolytic cleavage may have an N-terminal or C-terminal truncation of at least about 30 amino acids, at least about 40 amino acids, at least about 50, at least about 100 amino acids, about 120, about 130, approximately 140, approximately 150, approximately 160, approximately 170, approximately 180, approximately 190, approximately 200, approximately 210, approximately 220, approximately 230, approximately 240, approximately 250, approximately 275, approximately 300, approximately 350, approximately 400, about 450, about 500, or about 550 amino acids relative to SEQ ID NO: 2, 3, 4, 5, 6, or 7. In some embodiments, the fragments that are included herein are formed by removing the C-terminal crystallization domain, for example, by proteolysis or by inserting a stop codon into the coding sequence. In some embodiments, the fragments that are included herein are formed by removal of the N-terminal signal peptide. N-terminal truncations may further include a methionine residue at the N-terminus.

Preferred pesticidal proteins of the present invention are encoded by a nucleotide sequence that is sufficiently identical to the nucleotide sequence of SEQ ID NO: 1-4, or pesticidal proteins are sufficiently identical to the amino acid sequence set forth in SEQ ID NO: 5-26. By the expression sufficiently identical is meant an amino acid or nucleotide sequence that has at least about 60 or 65% sequence identity, about 70 or 75% sequence identity, about 80% or 85% sequence identity, about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or higher sequence identity than the reference sequence, as shown using one of the alignment programs described in this document using dard parameters. One skilled in the art will understand that these values can be appropriately adjusted to determine the corresponding identity of the proteins that are encoded by the two nucleotide sequences, taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.

To determine the percent identity of two amino acid sequences or two nucleic acids, the sequences are aligned for optimal comparison. The percent identity between two sequences is a function of the number of identical positions common to the sequences (i.e., percent identity = number of identical positions / total number of positions (e.g., overlapping positions) x 100). In one embodiment, the two sequences are the same length. In another embodiment, the percent identity is calculated over the entire length of the reference sequence (i.e., the sequence disclosed herein as any of the sequences of SEQ ID NO: 1-26). The percentage of identity between two sequences can be determined using techniques similar to those described below, allowing gaps in the sequence or not allowing them. When calculating percent identity, the number of exact matches is usually calculated. Skip i.e. an alignment position in which a residue is present in one sequence but not in another is considered as a position with non-identical residues.

Determining the percent identity between two sequences can be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm used to compare two sequences is the algorithm cited by Karlin and Altschul (1990) Proc. Natl. Acad Sci. USA 87: 2264, modified as published by Karlin and Altschul (1993) Proc. Natl. Acad Sci. USA 90: 5873-5877. Such an algorithm is embedded in the BLASTN and BLASTX programs by Altschul et al. (1990) J. Mol. Biol. 215: 403. BLAST nucleotide searches can be performed using the BLASTN program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the pesticide-like nucleic acid molecules of the present invention. BLAST protein searches can be done using the program

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BLASTX, score = 50, word length = 3, to obtain amino acid sequences homologous to the molecules of the pesticidal proteins of the present invention. To obtain alignments with gaps for comparison purposes, the Gapped BLAST program (in BLAST 2.0) can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389. Alternatively, PSI-Blast can be used to conduct a second search that detects distant bonds between molecules. See Altschul et al. (1997) supra. When working with BLAST, Gapped BLAST, and PSIBlast programs, the default parameters set for the respective programs (for example, BLASTX and BLASTN) can be applied. Alignment can also be done manually by reconciliation.

Another non-limiting example of a mathematical algorithm used to compare sequences is the ClustalW algorithm (Higgins et al. (1994) Nucleic Acids Res. 22: 4673-4680). ClustalW compares the sequences and aligns the entire length of the amino acid or DNA sequence and thus can provide conservative data for the entire amino acid sequence. The ClustalW algorithm is used in several commercially available DNA / amino acid analysis software packages, such as the ALIGNX module included in the Vector NTI software package (Invitrogen Corporation, Carlsbad, CA). After alignment of amino acid sequences using the ClustalW program, the percentage of amino acid identity can be estimated. A non-limiting example of computer programs that are used to analyze ClustalW alignments includes GENEDOC ™. GENEDOC ™ (Karl Nicholas) allows you to evaluate the similarity and identity of amino acids (or DNA) in numerous proteins. Another non-limiting example of the mathematical algorithm used to compare the sequence is the algorithm Myers and Miller (1988) CABIOS 4: 11-17. Such an algorithm is integrated into the ALIGN program (version 2.0), which is part of the GCG Wisconsin Genetics software package, Version 10 (available from Accelrys, Inc., 9685 Scranton Rd., San Diego, California, USA). When working with the ALIGN program, the PAM120 residue substitution table, a penalty for continuing the gap equal to 12, and a penalty for opening a gap equal to 4 can be used to compare amino acid sequences.

Unless otherwise specified, GAP Version 10, which uses the algorithm described by Needleman and Wunsch (1970) J. Mol. Biol. 48 (3): 443-453 will be used to determine sequence identity or similarity using the following parameters:% identity and% similarity for the nucleotide sequence using a penalty for opening a gap of 50 and a penalty for continuation of 3 and a matrix leveling weights nwsgapdna.cmp; % identity or% similarity for the amino acid sequence using a penalty for opening a gap of 8, and a penalty for continuing equal to 2, and the program BLOSUM62 scoring program. Equivalent programs may also be used. By an equivalent program is meant any sequence comparison program that, for any two sequences to be compared, performs alignment having identical nucleotide residue matches and identical percent sequence identity compared to the corresponding alignment obtained using GAP Version 10.

The present invention also encompasses variants of nucleic acid molecules. Variants of nucleotide sequences encoding pesticidal proteins include those sequences that encode pesticidal proteins disclosed herein, but which differ conservatively due to the degeneracy of the genetic code, as well as those sequences that are sufficiently identical, as discussed above. Allelic variants that occur in vivo can be identified using well-known molecular biological techniques, such as polymerase chain reaction (PCR) and hybridization techniques, which are described below. Variants of nucleotide sequences also include synthesized nucleotide sequences that were created, for example, using site-specific mutagenesis a, but which still encode the pesticidal proteins disclosed herein, as discussed below. Variants of the proteins that are included in the present invention are biologically active, which means that they continue to exhibit the desired biological activity of the native protein, that is, pesticidal activity. By the expression “retains activity” is meant that the variant will possess at least about 30%, at least about 50%, at least about 70%, or at least about 80% of the pesticidal activity of the native protein. Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83: 24802485; Andrews et al. (1988) Biochem. J. 252: 199-206; Marrone et al. (1985) J. of Economic Entomology 78: 290-293; and US Patent No. 5743477, all of which are incorporated herein by reference in their entirety.

An experienced specialist will also understand that changes can be made due to mutations in the nucleotide sequences of the present invention, thereby leading to changes in the amino acid sequence encoding pesticidal proteins without changing the biological activity of the proteins. Therefore, variants of isolated nucleic acid molecules can be created

- 5 033842 by introducing one or more nucleotide substitutions, insertions or deletions in the corresponding nucleotide sequence disclosed herein, so that one or more substitutions, inserts or deletions of amino acids are introduced into the encoded protein. Mutations can be induced using standard techniques such as site-specific mutagenesis and PCR-mediated mutagenesis. Such nucleotide sequence variants are also included in the present invention.

For example, conservative amino acid substitutions can be made in one or more, predicted, replaceable amino acid residues. A replaceable amino acid residue is a residue that can be changed from the sequence of a wild-type pesticidal protein without changing biological activity, while an essential amino acid residue is necessary for biological activity. A conservative amino acid substitution is one if the amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been identified in the art. These families include amino acids with major side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, fe nilalanine, tryptophan, histidine).

Delta endotoxins typically have five conserved sequence domains and three conserved structural domains (see, for example, de Maagd et al. (2001) Trends Genetics 17: 193-199). The first conservative structural domain contains seven alpha helices and is associated with incorporation into the membrane and pore formation.

Domain II consists of three folded beta layers that are ordered in the Greek key structure, and domain III consists of two anti-parallel folded beta layers in a jelly-roll type structure (de Maagd et al., 2001, see above). Domains II and III are involved in the recognition and binding of receptors and, therefore, are considered to be determinants of toxin specificity.

Amino acid substitutions can be made in non-conservative sites that retain function. In general, such substitutions are not carried out for conserved amino acid residues, or for amino acid residues located within a conservative motif, where such residues are indispensable for protein activity. Examples of residues that are conservative and which may be essential for protein activity include, for example, residues that are identical for all proteins included in sequence alignment of similar or related toxins with respect to the sequence of the present invention (e.g., residues that are identical when aligning homologous proteins). Examples of residues that are conservative, but which can allow conservative amino acid substitutions while still maintaining activity, include, for example, residues that have only conservative substitutions for all proteins included in the alignment of sequences of similar or related toxins with respect to the sequence of the present invention ( for example, residues that have only conservative substitutions for all homologous proteins included in the alignment). However, one skilled in the art will understand that functional variants may have slight conservative or non-conservative differences in conserved residues.

Alternatively, variants of the nucleotide sequences can be obtained by inducing mutations randomly along the entire length or part of the coding sequence, such as using saturating mutagenesis, and the resulting mutants can be screened for their ability to provide pesticidal activity to detect mutants. retaining activity. After the induction of mutagenesis, the encoded protein can be expressed recombinantly, and the activity of this protein can be determined using standard analysis techniques.

Using methods such as PCR, hybridization, etc., it is possible to identify the corresponding pesticidal sequences, such sequences that have significant identity with the sequence of the present invention. See, for example, Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and Innis, et al. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, NY).

Using the hybridization method, all or part of the pesticidal nucleotide sequence can be used to screen cDNA or genomic libraries. Methods for constructing such cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook and Russell, 2001, supra. So-called hybridization probes can be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and they can be labeled with a detectable group, such as 32P, or any other detectable marker, such as other radioactive isotopes, fluorescent compound, enzyme or enzyme cofactor. Probes

- 6,033,842 for hybridization can be obtained by labeling synthetic oligonucleotides based on the known nucleotide sequence encoding the pesticidal protein disclosed herein. Additionally, degenerate primers designed based on conserved nucleotides or amino acid residues in a nucleotide sequence or encoded amino acid sequence can be used. The probe typically contains a portion of the nucleotide sequence that hybridizes under stringent conditions with at least about 12, at least about 25, at least about 50, 75, 100, 125, 150, 175 or 200 consecutive nucleotides of the nucleotide sequence encoding the pesticidal protein the present invention or a fragment or variant thereof. Methods for preparing hybridization probes are generally known in the art and are disclosed in Sambrook and Russell, 2001, supra, incorporated herein by reference.

For example, the entire pesticidal sequence disclosed herein, or one or more of its parts, can be used as a probe capable of specific hybridization with corresponding sequences like pesticidal protein sequences and messenger RNA molecules. To achieve specific hybridization under various conditions, such probes contain sequences that are unique and preferably have a length of at least about 10 nucleotides or at least about 20 nucleotides. Such probes can be used to amplify the corresponding pesticidal sequences from a selected organism using PCR. This technique can be used to isolate additional coding sequences from the desired organism or as a diagnostic analysis to determine the presence of the coding sequence in the body. Hybridization techniques include hybridization screening of DNA-seeded plates (plaques or colonies; see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).

Thus, the present invention includes probes for hybridization, as well as nucleotide sequences capable of hybridizing all or part of the nucleotide sequence of the present invention (for example, at least about 300 nucleotides in length, at least about 400, at least about 500, 1000, 1200 , 1500, 2000, 2500, 3000, 3500 or up to the full length of the nucleotide sequence disclosed herein). Hybridization of such sequences can be carried out under stringent conditions. Stringent conditions or stringent hybridization conditions mean the conditions under which the probe will hybridize with its target sequence to a higher detectable degree than with other sequences (for example, at least 2 times higher than the background level). Strict conditions are sequence dependent and will vary under different circumstances. By monitoring the stringency of the hybridization and / or washing conditions, target sequences that are 100% complementary to the probe (homologous probing) can be identified. Alternatively, stringency conditions can be adjusted to allow for inconsistencies in the sequence so that lower degrees of similarity can be detected (heterologous sounding). In general, the probe has a length of less than about 1000 nucleotides, preferably less than 500 nucleotides.

Typically, harsh conditions will be those in which the salt concentration is less than about 1.5 M Na ions, usually from about 0.01 to 1.0 M Na ions (or other salts) at a pH of from 7.0 to 8.3 and the temperature is at least about 30 ° C for short probes (for example, from 10 to 50 nucleotides) and at least about 60 ° C for long probes (for example, more than 50 nucleotides). Strict conditions can also be achieved by the addition of destabilizing agents such as formamide. Illustrative low stringency conditions include hybridization with a buffer solution containing 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37 ° C, and washing in SSC from 1X to 2X (20X SSC = 3.0 M NaCl / 0.3 M trisodium citrate) at 50-55 ° C. Illustrative medium stringency conditions include hybridization with a solution containing from 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37 ° C, and washing in SSC from 0.5X to 1X SSC at 55-60 ° C. Typical high stringency conditions include hybridization with a solution containing 50% formamide, 1 M NaCl, 1% SDS at 37 ° C, and washing in 0.1X SSC at 60-65 ° C. Optionally, wash buffers may include from about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, usually from about 4 to about 12 hours.

The specificity, as a rule, depends on the washings after hybridization, in which the most important factors are the ionic strength and temperature of the final solution for washing. For DNA DNA hybrids, the Tm value can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138: 267-284: Tm = 81.5 ° C + 16.6 (log M) + 0.41 (% GC) - 0.61 (% form) - 500 / L; where M is the molarity of monovalent cations,% GC is the percentage of guanosine-containing and cytosine-containing nucleotides in DNA,% form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in a base pair. Tm is the temperature (at a given ionic strength and pH) at which 50% of the complementary target sequence hybridizes to perfectly match

- 7 033842 with an existing probe. Tm decreases by about 1 ° C with a violation of compliance for every 1%; thus, Tm, hybridization and / or washing conditions can be adjusted to hybridize to sequences with a desired degree of identity. For example, when searching for sequences with> 90% identity, Tm can be reduced by 10 ° C. In general, stringent conditions are chosen so that the temperature is about 5 ° C below the melting point (Tm) for a particular sequence and its complementary chain at given values of ionic strength and pH. However, very stringent conditions can use hybridization and / or washing at a temperature of 1, 2, 3 or 4 ° C below the melting point (Tm); medium stringency conditions may use hybridization and / or washing at a temperature of 6, 7, 8, 9, or 10 ° C below the melting point (Tm); low stringency conditions may use hybridization and / or washing at a temperature of 11, 12, 13, 14, 15, or 20 ° C. below the melting point (Tm). Using the equation, hybridization and washing compositions and the required Tm, one of ordinary skill in the art will understand that variations in the severity of hybridization conditions and / or washing solutions are essentially described. If the required degree of mismatch results in a Tm of less than 45 ° C (aqueous solution) or 32 ° C (formamide solution), then it is preferable to increase the concentration so that a higher temperature can be used. Detailed guidance on nucleic acid hybridization is given in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). Cm. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).

Isolated proteins and their variants and fragments

Pesticidal proteins are also encompassed in the present invention. By pesticidal protein is meant a protein having the amino acid sequence set forth in SEQ ID NO: 5-26. Their fragments, biologically active parts and variants are also presented and can be used to put into practice the methods of the present invention. The expression isolated protein or recombinant protein is used in relation to a protein that is no longer in its natural environment, but is located, for example, in an in vitro system or in a recombinant bacterial or plant host cell.

Fragments or biologically active parts include fragments of a polypeptide that contains amino acid sequences that are sufficiently identical to the amino acid sequence set forth in SEQ ID NO: 5-26 and exhibit pesticidal activity. The biologically active part of the pesticidal protein may be a polypeptide of length, for example, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350 or more amino acids. Such biologically active parts can be prepared using recombinant techniques and evaluated for pesticidal activity. Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83: 2480-2485; Andrews et al. (1988) Biochem. J. 252: 199-206; Marrone et al. (1985) J. of Economic Entomology 78: 290-293; and US patent No. 5743477, all of which are incorporated herein by reference in their entirety. As used herein, a fragment contains at least 8 contiguous amino acids from SEQ ID NO: 5-26. However, the present invention encompasses other fragments, such as any fragment in a protein, longer than about 10, 20, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350 or more amino acids.

By variants are meant proteins or polypeptides having an amino acid sequence that is at least about 60, 65%, about 70, 75%, about 80, 85%, about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% is identical to the amino acid sequence of any of the sequences of SEQ ID NO: 5-26. Options also include polypeptides encoded by a nucleic acid molecule that hybridizes with the nucleic acid molecule of SEQ ID NO: 1-4 or its complementary strand under stringent conditions. Options include polypeptides that differ in amino acid sequences as a result of mutagenesis. Variants of the proteins that are encompassed in the present invention are biologically active, that is, they continue to maintain the desired biological activity of the native protein, that is, retain pesticidal activity. In some embodiments, the variants have improved activity relative to the native protein. Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83: 2480-2485; Andrews et al. (1988) Biochem. J. 252: 199-206; Marrone et al. (1985) J. of Economic Entomology 78: 290-293; and US Patent No. 5,743,477, all of which are incorporated herein by reference in their entirety.

Bacterial genes, such as the axmi genes of the present invention, quite often have several methionine initiator codons located close to the beginning of the open reading frame. Often, translation initiation in one or more of these start codons will result in the formation of a functional protein. These start codons may include ATG

- 8 033842 codons. However, bacteria such as Bacillus sp. GTG codon will also be recognized as a start codon, and proteins that initiate translation in GTG codons contain methionine as the first amino acid. In rare cases, translation in bacterial systems can begin at the TTG codon, although in this case, TTG encodes methionine. In addition, it is often not initially determined which of these codons is used in vivo in bacteria. Thus, it is understood that the use of one of the alternative methionine codons can also lead to the creation of pesticidal proteins. These pesticidal proteins are encompassed in the present invention and can be used in the methods of the present invention. It will be understood that when expressed in plants, it will be necessary to change the alternative start codon to ATG in order to transmit accordingly.

In various embodiments of the present invention, pesticidal proteins comprise sequences derived from full length nucleotide sequences disclosed herein and amino acid sequences that are shorter than full length sequences by using the downstream alternative translation start site. Thus, the nucleotide sequence of the present invention and / or vectors, host cells and plants containing the nucleotide sequence of the present invention (and methods for producing and using the nucleotide sequence of the present invention) may contain a nucleotide sequence that encodes an amino acid sequence corresponding to SEQ ID NO: 6 , 7, 8, 9, 10, 12, 13, 14, 16, 17, 18, 19, 21, 22, 23, 24, 25, and 26.

Antibodies to the polypeptides of the present invention or to variants or fragments thereof are also encompassed. Methods for producing antibodies are well known in the art (see, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; US Patent No. 4196265).

Thus, one aspect of the present invention relates to antibodies, single chain antigen binding molecules or other proteins that specifically bind to one or more of the proteins or peptide molecules of the present invention and their homologues, fusions or fragments. In a particularly preferred embodiment, the antibody specifically binds to a protein having the amino acid sequence set forth in SEQ ID NO: 5-26, or a fragment thereof. In another embodiment, the antibody specifically binds to a fusion protein containing an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NO: 5-26, or a fragment thereof.

The antibodies of the present invention can be used to quantitatively or qualitatively determine the content of a protein or peptide molecule of the present invention or to detect post-translational modifications of proteins. As used herein, an antibody or peptide is said to specifically bind to a protein or peptide molecule of the present invention if such binding is not completely inhibited by the presence of unrelated molecules.

Antibodies of the present invention may be contained in a kit used to detect the protein or peptide molecules of the present invention. The invention further encompasses a method for detecting a protein or peptide molecule of the present invention (in particular, a protein encoded by the amino acid sequence set forth in SEQ ID NO: 5-26, including variants or fragments thereof capable of specifically binding to an antibody of the invention), comprising contacting the sample with an antibody of the invention and determining whether the sample contains a protein or peptide molecule of the invention. A method of using antibodies to detect a protein or peptide of interest is known in the art.

Modified or improved options

It is known that the DNA sequences of a pesticidal protein can be changed using various methods, and that these changes can lead to the formation of a DNA sequence encoding proteins with amino acid sequences different from those encoding the pesticidal proteins of the present invention. This protein can be modified in various ways, including amino acid substitutions, deletions, truncation and insertion of one or more amino acid sequences from SEQ ID NO: 5-26, including up to about 2, about 3, about 4, about 5, about 6, about 7 approximately 8, approximately 9, approximately 10, approximately 15, approximately 20, approximately 25, approximately 30, approximately 35, approximately 40, approximately 45, approximately 50, approximately 55, approximately 60, approximately 65, approximately 70, approximately 75, approximately 80, approximately 85, approximately 90, approximately 100, approximately 105, approximately 110, approximately 115, approximately 120, approximately 125, about 130, about 135, about 140, about 145, about 150, about 155 or more amino acid substitutions, deletions or insertions. Methods for carrying out such manipulations are generally known in the art. For example, amino acid sequence variants of a pesticidal protein can be obtained using mutations in DNA. This can also be achieved using one of several varieties of mutagenesis and / or by directed development. In some aspects, changes encoded by amino acid sequences will not have a significant effect on protein function. Such options will have the desired pesticidal activity. However, it is understood that the ability of a pesticidal protein to provide pesticidal activity can be improved by

- 9,033,842 applying such techniques to the compositions of the present invention. For example, a pesticidal protein can be expressed in host cells that exhibit high levels of base insertion errors in DNA replication, such as XL-1 Red (Stratagene, La Jolla, CA). After propagation in such strains, DNA can be isolated (for example, by obtaining plasmid DNA or by amplification by PCR and cloning the resulting PCR fragment into a vector), cultivate the pesticidal protein with mutations in the non-mutagenic strain, and identify mutated genes with the pesticidal activity, for example, by conducting an analysis to study pesticidal activity. In general, protein is mixed and used in feeding analyzes. See, for example, Marrone et al. (1985) J. of Economic Entomology 78: 290-293. Such assays may include bringing the plant into contact with one or more pests and determining the plant's ability to survive and / or the ability to cause pest death. Examples of mutations that lead to increased toxicity are found in Schnepf et al. (1998) Microbiol. Mol. Biol. Rev. 62: 775-806.

Alternatively, changes can be made to the sequence of many proteins at the amino or carboxy terminus, without significantly affecting the activity. Such changes may include insertions, deletions or changes induced using modern molecular methods, such as PCR, including PCR amplification, which alter or extend the protein coding sequence by inserting amino acid coding sequences into the oligonucleotides used in PCR amplification. Alternatively, the added protein sequences may contain whole sequences encoding a protein, such as those commonly used in the art to produce fusion proteins. Such fusion proteins are often used (1) to increase the expression of a protein of interest, (2) the introduction of a binding domain, enzymatic activity or epitope in order to facilitate protein purification, protein detection, or for other experimental uses known in the art, (3) the direction of secretion or translation of the protein into the subcellular organelle, such as the periplasmic space of gram-negative bacteria, the endoplasmic reticulum of eukaryotic cells, the latter often with Odita to protein glycosylation.

Variants of the nucleotide and amino acid sequences of the present invention also encompass sequences obtained using procedures leading to recombination and causing the formation of mutations, such as rearrangement in DNA. When performing such a technique, one or more different sites encoding pesticidal proteins can be used to create a new pesticidal protein with the desired properties. Thus, recombinant polynucleotide libraries are created from a population of related polynucleotide sequences that contain portions of a sequence that is substantially identical and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest can be rearranged between a pesticidal activity gene of the present invention and other known pesticidal genes to obtain a new gene encoding an interest protein sequence with an improved property, such as with increased insecticidal activity. Strategies for such a rearrangement in DNA are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad Sci. USA 91: 10747-10751; Stemmer (1994) Nature 370: 389-391; Crameri et al. (1997) Nature Biotech. 15: 436-438; Moore et al. (1997) J. Mol. Biol. 272: 336-347; Zhang et al. (1997) Proc. Natl. Acad Sci. USA 94: 4504-4509; Crameri et al. (1998) Nature 391: 288-291; and US patents No. 5605793 and No. 5837458.

Domain transfer or rearrangement is another mechanism for creating altered pesticidal proteins. Domains can move between pesticidal proteins, leading to the formation of hybrid or chimeric toxins with improved pesticidal activity or various target characteristics. Methods for creating recombinant proteins and their study regarding pesticidal activity are well known in the art (see, for example, Naimov et al. (2001) Appl. Environ. Microbiol. 67: 5328-5330; de Maagd et al. (1996) Appl. Environ. Mcrobiol. 62: 1537-1543; Ge et al. (1991) J. Biol. Chem. 266: 17954-17958; Schnepf et al. (1990) J. Biol. Chem. 265: 20923-20930; Rang et al. 91999) Appl. Environ. Microbiol. 65: 2918-2925).

Vectors

The pesticidal sequence of the present invention may be presented in an expression cassette for expression in a plant of interest. By plant expression cassette is meant a DNA construct that is capable of expressing a protein from an open reading frame in a plant cell. Usually they contain a promoter and a coding sequence. Often such designs will also contain a 3'-untranslated portion. Such constructs may contain a signal sequence or leader sequence to facilitate co-translational or post-translational transport of the peptide to specific intracellular structures, such as chloroplast (or other plastid), endoplasmic reticulum or Golgi complex.

- 10 033842

By signal sequence is meant a sequence for which it is known or assumed to lead to co-translational or post-translational transport of peptides across the cell membrane. In eukaryotes, it is usually associated with secretion in the Golgi apparatus, with some degree of resulting glycosylation. Insecticidal bacterial toxins are often synthesized as protoxins that are proteolytically activated in the intestines of the target insect (Chang (1987) Methods Enzymol. 153: 507-516). In some embodiments, the signal sequence is localized in the native sequence, or can be obtained from the sequence of the present invention. By leader sequence is meant any sequence that, when translated, leads to the formation of an amino acid sequence sufficient to trigger cotranslational transport of the peptide chain to the subcellular organelle. Thus, leader sequences include leader sequences that have a targeted effect on transport and / or glycosylation due to passage into the endoplasmic reticulum, passage into vacuoles, plastids, including chloroplasts, mitochondria, and the like.

By plant transformation vector is meant a DNA molecule that is necessary for the efficient transformation of a plant cell. Such a molecule may contain one or more plant expression cassettes and may be organized into more than one vector DNA molecule. For example, binary vectors are plant transformation vectors that use two non-contiguous DNA vectors to encode all the required functions with activity at cis and trans positions to transform plant cells (Hellens and Mullineaux (2000) Trends in Plant Science 5: 446- 451). The expression vector refers to a nucleic acid construct intended for transfer between different host cells. An expression vector is a vector that has the ability to introduce, integrate and express heterologous DNA sequences or fragments thereof in a foreign cell. The cassette will comprise 5 'and / or 3' regulatory sequences functionally linked to the sequence of the present invention. By functionally linked is meant a functional linkage between a promoter sequence and a second sequence, where the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. In general, the expression functionally linked means that the linked nucleotide sequences are adjacent and, in cases where it is necessary to combine two sections encoding a protein, they are adjacent and are in the same reading frame. A cassette may further comprise at least one additional gene to be co-transformed into an organism. Alternatively, the additional gene (s) can be delivered by a variety of expression cassettes.

In various embodiments, the nucleotide sequence of the present invention is operably linked to a promoter, for example, a plant promoter. A promoter is a nucleotide sequence whose function is to control the transcription of the underlying coding sequence. A promoter, along with other transcriptional and translational regulatory nucleotide sequences (also called control sequences), are necessary for the expression of the DNA sequence of interest.

Such an expression cassette is provided by a plurality of restriction sites for insertion of the pesticidal sequence to be transcriptionally regulated by regulatory sites.

The expression cassette will include, in the 5'-3 'direction of transcription, a transcription and translation initiation site (i.e., a promoter), a DNA sequence of the present invention and a translation and transcription termination site (i.e., a termination site), functioning in a plant. The promoter may be native or similar, or may be foreign or heterologous to the host plant and / or to the DNA sequence of the present invention. Additionally, the promoter may be a natural sequence or, alternatively, a synthetic sequence. Where the promoter is native or homologous to the host plant, it is understood that the promoter is found in the native plant into which the promoter is introduced. Where the promoter is foreign or heterologous to the DNA sequence of the present invention, it is understood that the promoter is not native or naturally occurring for the functionally linked DNA sequence of the present invention.

The expression site may be native to the site of transcription initiation, may be native to a functionally linked DNA sequence of interest, may be native to the host plant, or may be obtained from another source (i.e., foreign or heterologous to the promoter , a DNA sequence of interest to a host plant or any combination thereof). Suitable termination sites can be obtained from A. tumefaciens Ti plasmids, such as octopinsynthase and nopaline synthase termination sites. See also Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64: 671-674; Sanfacon et al.

- 11,033,842 (1991) Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91: 151158; Ballas et al. (1989) Nucleic Acids Res. 17: 7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15: 96279639.

Where required, the gene (s) can be optimized to increase expression in the transformed host cell. Thus, genes can be synthesized using codons preferred for the host cell to improve expression, or can be synthesized using codons at a frequency of use of codons preferred for the host cell. In general, the content of GC in the gene will increase. See, for example, Campbell and Gowri (1990) Plant Physiol. 92: 1-11, which discusses the use of codons preferred by the host cell. In the art there are methods for synthesizing plant preferred genes. See, for example, US Pat. Nos. 5,380,831 and 5,436,391, US Patent Publication No. 20090137409 and Murray et al. (1989) Nucleic Acids Res. 17: 477-498, which are incorporated herein by reference.

In one embodiment, the pesticidal protein is sent to the chloroplast for expression. Thus, in cases where the pesticidal protein is not directly introduced into the chloroplast, the expression cassette will additionally contain a nucleic acid encoding a transit peptide that directs the pesticidal protein into the chloroplasts. Such transit peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J. Biol. Chem. 264: 17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84: 965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196: 1414-1421; and Shah et al. (1986) Science 233: 478-481.

The gene that determines the pesticidal activity that should be directed to the chloroplast can be optimized for expression in the chloroplast to take into account the differences in the use of the codon in the plant nucleus and this organelle. Thus, nucleic acids of interest can be synthesized using codons preferred for chloroplasts. See, for example, US patent No. 5380831, which is incorporated herein by reference.

Plant transformation

The methods of the present invention include introducing a nucleotide construct into a plant. By introduction is meant the provision of a plant with a nucleotide construct so that the construct gains access to the interior of the plant cell. The methods of the present invention do not require the use of a specific method of introducing a nucleotide construct into a plant, but only so that the nucleotide construct has access to the interior of at least one plant cell. Methods for introducing nucleotide constructs into plants are known in the art, including but not limited to methods for stable transformation, methods for temporary transformation, and methods mediated by viruses.

By plant is meant whole plants, plant organs (for example, leaves, stems, roots, etc.), seeds, plant cells, shoots, embryos and offspring of these plants. Plant cells can be differentiated or undifferentiated (for example, callus, cell culture suspension, protoplasts, leaf cells, root cells, phloem cells, pollen).

Transgenic plants or transformed plants or stably transformed plants or cells or tissues refer to plants that have exogenous nucleotide sequences or DNA fragments introduced or integrated into a plant cell. These nucleotide sequences incorporate those that are exogenous or not present in an untransformed plant cell, as well as those that may be endogenous, or present in an untransformed plant cell. The heterologous expression generally refers to nucleotide sequences that are not endogenous to the cell or are not part of the native genome in which they are present, and which were introduced into the cell by infection, transfection, microinjection, electroporation, microparticle bombardment, etc. .

The transgenic plants of the present invention express one or more of the new toxin sequences disclosed herein. In various embodiments, the transgenic plant further comprises one or more additional insect resistance genes (e.g., Cry1, such as members of the Cry1A, Cry1B, Cry1C, Cry1D, Cry1E and Cry1F families; Cry2, such as a representative of the Cry2A family; Cry9, such as representatives families Cry9A, Oy9B, Cry9C, Cry9D, Cry9F and Cry9F, etc.). One skilled in the art will understand that a transgenic plant may contain any gene that reports an agricultural trait of interest.

Transformation of plant cells can be carried out using one or more methods known in the art. The gene for determining pesticidal activity of the present invention can be modified to obtain or increase expression in plant cells. Typically, a construct that expresses such a protein contains a promoter that regulates gene transcription, as well as a 3 'untranslated region that provides transcription termination and polyadenylation. The structure of such constructs is well known in the art. In some cases, it may be appropriate to construct the gene in such a way that

- 12,033,842 will result in secretion of the resulting peptide or other targeted action in the plant cell. For example, a gene can be designed to contain a signal peptide to facilitate transfer of the peptide to the endoplasmic reticulum. It may also be preferable to design an intron-containing expression cassette in such a way that the mRNA processing of this intron is required for expression.

Typically, a given plant expression cassette will be introduced into a plant transforming vector. This plant transforming vector may contain one or more DNA vectors necessary to achieve plant transformation. For example, it is common practice in the art to use plant transforming vectors that contain more than one adjacent DNA segment. In the art, these vectors are often referred to as binary vectors. Binary vectors, as well as vectors with helper plasmids, are most often used for Agrobacterium-mediated transformation, where the size and total length of the DNA segments required to achieve efficient transformation are large enough, and it is advisable to separate functions by individual DNA molecules. Binary vectors, as a rule, contain a plasmid vector that contains cis-acting sequences necessary for T-DNA transfer (such as those located on the left border and on the right border of the fragment), a selectable marker that is designed to be capable of expression in a plant cell, and a gene of interest (a gene designed in such a way as to be capable of expression in a plant cell, which requires the creation of transgenic plants). Also in this plasmid vector there are sequences necessary for the replication of bacteria. The cis-acting sequences are organized in such a way as to ensure efficient transfer into and expression of plant cells. For example, a selectable marker gene and a gene that determines pesticidal activity are located between the left and right borders. Often the second plasmid vector contains trans-acting factors that mediate T-DNA transfer from Agrobacterium to plant cells. The plasmid often contains virulence genes (Vir genes) that allow infection of plant cells of Agrobacterium and DNA transfer by cleaving in the region of border sequences and vir-mediated DNA transfer, as is known in the art (Hellens and Mullineaux (2000) Trends in Plant Science 5 : 446-451). Several types of Agrobacterium strains (e.g., LBA4404, GV3101, EHA101, EHA105, etc.) can be used to transform plants. The second plasmid vector is not necessary for plant transformation using other methods, such as microparticle bombardment, microinjection, electroporation, transfection with polyethylene glycol, etc.

In general, plant transformation methods include transferring heterologous DNA to target plant cells (e.g., immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by appropriate selection with a maximum threshold level of appropriate selection (in depending on the selectable marker gene) in order to isolate the transformed plant cells from the group of non-transformed cell mass. Explants are usually transferred to a fresh portion of the same culture medium and cultured standardly. Next, the transformed cells differentiate into shoots after placing them on a regenerative medium, into which a means for selection at a concentration of the maximum threshold level is added. The shoots are then transferred to a selective substrate for rooting plants to root the shoot or seedling. The transgenic seedling then grows into a mature plant and produces fertile seeds (e.g., Hiei et al. (1994) The Plant Journal 6: 271-282; Ishida et al. (1996) Nature Biotechnology 14: 745-750). Explants, as a rule, are transferred to a fresh portion of the same culture medium and cultured as standard. A general description of the techniques and methods for producing transgenic plants is given in Ayres and Park (1994) Critical Reviews in Plant Science 13: 219-239 and Bommineni and Jauhar (1997) Maydica 42: 107-120. Because the transformed material contains numerous cells; both transformed and non-transformed cells are present in any part of the treated target callus or tissue or group of cells. The ability to cause the death of untransformed cells and to allow the proliferation of transformed cells allows to obtain cultures of transformed plants. Often, the ability to remove non-transformed cells is a limitation for the rapid isolation of transformed plant cells and the successful creation of transgenic plants.

Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocotyledonous or dicotyledonous, intended for transformation. The creation of transgenic plants can be carried out using one of several methods, including, but not limited to, microinjection, electroporation, directed gene transfer, introduction of heterologous DNA using Agrobacterium into plant cells (Agrobacterium-mediated transformation), bombardment of plant cells with heterologous foreign-conjugated particles DNA, accelerated particle method, transformation using an aerosol beam injector (published US patent application

- 13 033842 No. 20010026941; US patent No. 4945050; international publication No. WO 91/00915; U.S. Patent Application Publication No. 2002015066), Hse1 Transformation and various other DNA transfer methods not mediated by direct particle administration.

Methods for transforming chloroplasts are known in the art. See, for example, Svab ct al. (1990) Proc. Natl. Acad Sei. USA 87: 8526-8530; Svab and Maliga (1993) Proc. Natl. Acad Sei. USA 90: 913-917; Svab and Maliga (1993) EMBO J. 12: 601-606. This method is based on the use of a gene gun for the delivery of DNA containing a selectable marker and the targeted introduction of DNA into the plastid genome through homologous recombination. Additionally, the transformation of plastids can be achieved by trans-activation of a silent, plastid transgene due to tissue-preferred expression of plastid, nuclear RNA polymerase. Such a system is described in McBridc ct al. (1994) Proc. Natl. Acad Sci. USA91: 7301-7305.

After integration of heterologous foreign DNA into plant cells, appropriate selection is used with a maximum threshold level of appropriate selection in the medium to cause the death of untransformed cells and to separate, as well as to proliferate, the allegedly transformed cells that survive as a result of this selection treatment, regularly transferring the cells to fresh Wednesday Through continuous passage and administration of an appropriate selection agent, the proliferation of cells that have been transformed with the plasmid vector is identified and stimulated. Molecular and biochemical methods can then be used to confirm the presence of an integrated heterologous gene of interest in the genome of the transgenic plant.

Cells that have been transformed can be grown into plants in accordance with conventional methods. See, for example, McCormick ct al. (1986) Plant Cell Reports 5: 81-84. These plants can then be grown and either pollinated with the same transformed line or another line, and the resulting hybrid, which has constitutive expression of the desired phenotypic characteristic, can be identified. Two or more generations can be grown in order to make sure that the expression of the desired phenotypic trait is stably preserved and inherited, and then the seeds are harvested to make sure that the expression of the desired phenotypic trait has been achieved. Thus, the present invention provides transformed seeds (also called transgenic seeds) containing the nucleotide construct of the present invention, for example, the expression cassette of the present invention, stably integrated into their genome.

Plant Transformation Assessment

After the introduction of heterologous foreign DNA into plant cells, the transformation or integration of the heterologous gene into the plant genome is confirmed in various ways, such as analysis of nucleic acids, proteins and metabolites associated with the integrated gene.

PCR analysis is an express technique for screening transformed cells, tissue or shoots for the presence of an introduced gene at an earlier stage before transplanting into the soil (Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor , NY). PCR is performed using oligonucleotide primers specific for the gene of interest or the background of the Agrobacterium vector, etc.

Plant transformation can be confirmed using Southern blot analysis of genomic DNA (Sambrook and Russell, 2001, supra). In general, total DNA is extracted from the transformant, digested with the appropriate restriction enzymes, fractionated on an agarose gel and transferred onto a nitrocellulose or nylon membrane. The membrane or blot is then examined using, for example, a ³² P radiolabeled target DNA fragment to confirm the integration of the introduced gene into the plant genome according to standard techniques (Sambrook and Russell, 2001, supra).

During the Northern blot analysis, RNA was isolated from special transformant tissues, fractionated on an agarose gel containing formaldehyde, and transferred to a nylon filter according to standard methods that are regularly used in the art (Sambrook and Russell, 2001, supra). The RNA expression encoded by the pesticidal activity determining gene is then examined by hybridization on a filter with a radioactive probe obtained from the pesticidal gene using methods known in the art (Sambrook and Russell, 2001, supra).

Confirmation of the presence of a protein encoded by a gene that determines pesticidal activity can be carried out on transgenic plants using standard Western blot procedures, biochemical analyzes and the like on transgenic plants (Sambrook and Russell, 2001, supra) using antibodies that bind to one or several epitopes present on a pesticidal protein.

Pesticidal activity in plants

In another aspect of the present invention, transgenic plants can be created in which a pesticidal protein that has pesticidal activity is expressed. To create transgenic plants, you can use the methods described above as an example, but the method by which transgenic plant cells are obtained is not critical to the present invention.

- 14,033,842 tenias. At the discretion of the experimenter, methods known or described in the art can be applied, such as Agrobacterium-mediated transformation, biolistic transformation and non-particle mediated methods. Plants expressing the pesticidal protein can be isolated by well-known methods described in the art, for example, by transforming callus, selecting transformed callus and regenerating the fruiting plant from such transgenic callus. During this method, any gene can be used as a selectable marker, provided that its expression in plant cells provides the opportunity for identification or selection of transformed cells.

A variety of markers have been developed for use in plant cells, such as resistance to chloramphenicol, aminoglycoside G418, hygromycin, or the like. Other genes that encode a product involved in chloroplast metabolism can also be used as selectable markers. For example, genes that provide herbicide resistance to plants, such as glyphosate, bromoxynil or imidazolinone, may find particular use. Such genes have been described in the literature (Stalker et al. (1985) J. Biol. Chem. 263: 6310-6314 (bromoxynil nitrilase resistance gene); and Sathasivan et al. Acids Res. 18: 2188 (AHAS imidazolinone resistance gene) Additionally, the genes disclosed herein are useful as markers for assessing the transformation of bacterial or plant cells. Methods for detecting the presence of a transgene in a plant, plant organ (eg, leaves, stems, roots, etc.), seed, plant cell , sprout, germ or their offspring are well known in the art. In an embodiment, the presence of a transgene is detected by investigating pesticidal activity.

Fertile plants in which a pesticidal protein is expressed can be tested for pesticidal activity, and for further crossbreeding, selection of plants in which optimal activity is manifested can be carried out. Pesticidal activity assay methods are available in the art. Typically, protein is mixed and used in feeding assays. See, for example, Marrone et al. (1985) J. of Economic Entomology 78: 290-293.

The present invention can be used to transform any kind of plant, including, but not limited to, monocotyledonous and dicotyledonous plants. Examples of plants of interest include, but are not limited to, corn (maize), sorghum, wheat, sunflower, tomato, cruciferous, pepper, potato, cotton, rice, soybeans, sugar beets, sugarcane, tobacco, barley and oilseed rape, Brassica sp ., alfalfa, rye, millet, safflower, peanuts, sweet potato, cassava, coffee tree, coconut, pineapple, citrus trees, cocoa tree, tea bush, banana, avocado, fig tree, guava, mango tree, olive, papaya, cashews, macadamia, almonds, oatmeal, vegetables, ornamental plants and hvo nye.

Vegetables include, but are not limited to, tomatoes, lettuce, green beans, lima beans, peas, and members of the Curcumis genus, such as cucumber, cantaloupe, and cantaloupe. Ornamental plants include, but are not limited to, azalea, hydrangea, hibiscus, roses, tulips, yellow daffodils, petunia, cloves, poinsettia and chrysanthemum. Preferably, the plants of the present invention are cultivated plants (e.g., maize, sorghum, wheat, sunflower, tomato, cruciferous, pepper, potato, cotton, rice, soybeans, sugar beets, sugarcane, tobacco, barley, oilseed rape, etc. .).

Pest Control

General methods are known in the art for using strains containing the nucleotide sequence of the present invention or its variant as pesticidal agents in pest control or in the creation of other organisms. See, for example, US patent No. 5039523 and EP 0480762A2.

Bacillus strains containing the nucleotide sequence of the present invention or a variant thereof, or microorganisms that are genetically modified so as to contain the pesticidal activity determining gene of the present invention and protein, can be used to protect crops and products from pests. In one aspect of the present invention, whole, i.e. non-lysed cells of the body producing the toxin (pesticide) are treated with reagents that prolong the activity of the toxin produced in the cell when the cell is placed in the environment of the target pest (s).

Alternatively, the pesticide is obtained by introducing a gene that determines pesticidal activity into a host cell. Expression of the gene that determines pesticidal activity directly or indirectly leads to intracellular production and maintenance of the pesticide level. In one aspect of the present invention, these cells are then treated under conditions that prolong the activity of the toxin produced in the cell when the cell is placed in the environment of the target pest (s). The resulting product retains the toxicity characteristic of the toxin. From these naturally encapsulated pesticides, the preparation can then be formulated in accordance with traditional methods for introducing the target pest into the habitat, for example, soil, water and plant foliage. See, for example, EPA 0192319 and the references cited therein. Alternatively, it is possible to formulate the preparation from cells expressing the gene of the present invention, thus

- 15,033,842 zom to ensure that the resulting material is used as a pesticide.

The active ingredients of the present invention are usually applied in the form of compositions, and can be applied simultaneously or sequentially with other compounds to the crop area or plant to be treated. These compounds can be fertilizers, herbicides, cryoprotectants, surfactants, detergents, pesticidal soaps, oils used at rest, polymers and / or compositions from slowly released or biodegradable carriers that allow long-term dosage in the target area after a single making composition. They can also be selective herbicides, chemical insecticides, viricides, microbicides, amoebicides, pesticides, fungicides, bactericides, nematicides, molluscicides or mixtures of several of these drugs, if necessary, together with additional agriculturally acceptable carriers, surfactants or excipients application enhancers commonly used in the art. Suitable carriers and excipients may be solid or liquid and correspond to substances that are commonly used in formulation technology, for example, natural or reduced minerals, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers. Similarly, the formulations can be obtained in the form of edible baits, or they can be shaped into traps for pests in order to ensure that the target pest can be fed or a pesticidal composition gets inside it.

Methods for applying the active ingredient of the present invention or the agrochemical composition of the present invention, which contains at least one of the pesticidal proteins produced by the bacterial strains of the present invention, include applying to leaves, coating seeds and applying to the soil. The number of applications and the application rate depends on the intensity of infection with the corresponding pest.

The composition can be in the form of a powder, dust, pellets, granules, aerosol emulsions, colloids, solutions or the like, and can be obtained by such traditional methods as drying, lyophilization, homogenization, extraction, filtration, centrifugation, precipitation or concentration of cell culture, including polypeptide. In all such compositions that contain at least one such pesticidal polypeptide, the polypeptide may be present in a concentration (in mass fractions) of from about 1% to about 99%.

Using the methods of the present invention in this area, it is possible to kill or reduce the number of Lepidoptera, Hemoptera, Diptera or Coleoptera, or can be used prophylactically in the environment to prevent infection by a sensitive pest. Preferably, the pest swallows or contacts a pesticidally effective amount of the polypeptide. By pesticidally effective amount is meant an amount of a pesticide that is capable of causing the death of at least one pest or significantly limiting pest growth, nutrition, or normal physiological development. This amount will vary depending on factors such as, for example, specific target pests to be controlled, specific environment, location, plant, crop or agricultural plot to be treated, environmental conditions and method, rate, concentration, stability and amount of application a polypeptide composition providing a pesticidal effect. The compositions can also be modified taking into account climatic conditions, environmental impact and / or frequency of application and / or severity of infection by pests.

The described pesticidal compositions can be prepared by preparing the preparation either from a bacterial cell, a crystal and / or a suspension of spores, or from an isolated protein component with the desired agriculturally acceptable carrier. Compositions can be formulated prior to administration using appropriate methods, such as lyophilization, freeze-drying, drying, or in an aqueous vehicle, medium, or a suitable diluent, such as saline or other buffer. The formulated compositions may be in the form of dust or granular material, or suspension in oil (vegetable or mineral), or aqueous, or oil / water emulsions, or as a wettable powder, or in combination with any other carrier material suitable for use in areas of agriculture. Suitable agricultural vehicles may be solid or liquid, and they are well known in the art. The expression agriculturally acceptable carrier encompasses all excipients, inert components, dispersants, surfactants, tackifiers, binders, etc., which are commonly used in pesticide formulation technology; they are well known to those skilled in the art of formulating pesticidal preparations. These compositions can be mixed with one or more solid or liquid excipients and obtained in various ways, for example, by uniformly mixing, mixing and / or grinding the pesticidal composition with suitable excipients using traditional formulation techniques. Suitable formulations and methods of application are described in US Pat. No. 6,468,523, which is incorporated herein by reference.

- 16 033842

The pest includes, but is not limited to, insects, fungi, bacteria, nematodes, ticks, ticks and the like. Pest insects include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Loptera, Tropoptera, Tropoptera, Tropoptera, Diptera.

Squad Coleoptera includes suborders Adephaga and Polyphaga. Suborder Adephaga includes superfamily Caraboidea and Gyrinoidea, while suborder Polyphaga comprises superfamily Hydrophiloidea, Staphylinoidea, Cantharoidea, Cleroidea, Elateroidea, Dascilloidea, Dryopoidea, Byrrhoidea, Cucujoidea, Meloidea, Mordelloidea, Tenebrionoidea, Bostrichoidea, Scarabaeoidea, Cerambycoidea, Chrysomeloidea and Curculionoidea. The superfamily Caraboidea includes the families Cicindelidae, Carabidae and Dytiscidae. The superfamily Gyrinoidea includes the family Gyrinidae. The superfamily Hydrophiloidea includes the Hydrophilidae family. The superfamily Staphylinoidea includes the families Silphidae and Staphylinidae. The superfamily Cantharoidea includes the families Cantharidae and Lampyridae. The superfamily Cleroidea includes the families Cleridae and Dermestidae. The superfamily Elateroidea includes the families Elateridae and Buprestidae. The Cucujoidea superfamily includes the Coccinellidae family. The superfamily Meloidea includes the Meloidae family. The Tenebrionoidea superfamily includes the Tenebrionidae family. The superfamily Scarabaeoidea includes the families Passalidae and Scarabaeidae. The Cerambycoidea superfamily includes the Cerambycidae family. The Chrysomeloidea superfamily includes the Chrysomelidae family. The superfamily Curculionoidea includes the families Curculionidae and Scolytidae.

Squad Diptera includes sub-squads Nematocera, Brachycera and Cyclorrhapha. The suborder Nematocera includes the families Tipulidae, Psychodidae, Culicidae, Ceratopogonidae, Chironomidae, Simuliidae, Bibionidae, and Cecidomyiidae. The suborder Brachycera includes the families Stratiomyidae, Tabanidae, Therevidae, Asilidae, Mydidae, Bombyliidae, and Dolichopodidae. The Cyclorrhapha suborder includes Aschiza and Aschiza sections. The Aschiza section includes the families Phoridae, Syrphidae and Conopidae. The Aschiza section includes sub-sections of Acalyptratae and Calyptratae. The Acalyptratae section includes the families Otitidae, Tephritidae, Agromyzidae and Drosophilidae. The Calyptratae section includes the families Hippoboscidae, Oestridae, Tachinidae, Anthomyiidae, Muscidae, Calliphoridae and Sarcophagidae.

The order Lepidoptera includes the families Papilionidae, Pieridae, Lycaenidae, Nymphalidae, Danaidae, Satyridae, Hesperiidae, Sphingidae, Saturniidae, Geometridae, Arctiidae, Noctuidae, Lymantriidae, Sesiidae and Tineidae.

Nematodes include parasitic nematodes, such as nodule, cyst-forming and damaging nematodes, including Heterodera spp., Meloidogyne spp. and Globodera spp .; in particular, representatives of cyst-forming nematodes, including, but not limited to, Heterodera glycines (soybean cyst-forming nematode); Heterodera schachtii (beet cyst-forming nematode); Heterodera avenae (cereal cyst-forming nematode); and Globodera rostochiensis and Globodera pailida (potato cyst-forming nematodes). Damaging nematodes include Pratylenchus spp.

Semi-winged pests (which include species designated as Hemiptera, Homoptera, or Heteroptera) include, but are not limited to, Lygus spp., Such as Western meadow bugs (Lygus hesperus), meadow bugs (Lygus lineolaris), and alfalfa bugs (Lygus elisus); plant aphids such as green peach aphids (Myzus persicae), cotton aphids (Aphis gossypii), cherry or cherry aphids (Myzus cerasi), soy aphids (Aphis glycines Matsumura); dark cicadas (Nilaparvata lugens) and rice green cicadas (Nephotettix spp.); and shields, such as the green shield (Acrosternum hilare), the marble moth (Halyomorpha halys), the southern green shield (Nezara viridula), the rice shield (Oebalus pugnax), the red-footed shield (Pentatoma rufipes), the European shield (Rhaphigaster nebulosa and) Troilus luridus.

The pests of the present invention for main crops include: maize: Ostrinia nubilalis, European corn moth; Agrotis ipsilon, ipsilon scoop; Helicoverpa zea, corn scoop; Spodoptera frugiperda, grass scoop; Diatraea grandiosella, south-west corn moth; Elasmopalpus lignosellus, grain grinder; Diatraea saccharalis, sugarcane moth; Diabrotica virgifera, western corn root beetle; Diabrotica longicornis barberi, northern corn root beetle; Diabrotica undecimpunctata howardi, southern corn root beetle; Melanotus spp., Nutcracker beetles; Cyclocephala borealis, northern chrysanthemum (larva of the khrushchev); Cyclocephala immaculata, Southern Chrysanthemum (larva of the Khrushchev); Popillia japonica, Japanese chipper; Chaetocnema pulicaria, earthen corn flea; Sphenophorus maidis, corn weevil; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis, corn root aphid; Blissus leucopterus leucopterus, North American wheat bug; Melanoplus femurrubrum, red-legged filly; Melanoplus sanguinipes, migratory filly; Hylemya platura, sprout fly larva; Agromyza parvicornis, mottled corn moth; Anaphothrips obscrurus, cereal thrips; Solenopsis milesta, a thief ant; Tetranychus urticae, an ordinary spider mite; Sorghum: Chilo partellus, a sorghum grinder; Spodoptera frugiperda, grass scoop; Helicoverpa zea, corn scoop; Elasmopalpus lignosellus, a small corn stalk grinder; Feltia subterranea, winterworm caterpillar; Phyllophaga crinita, Maybug larva; Eleodes, Conoderus and Aeolus spp., Wireworms; Oulema melanopus, red-breasted drunkard; Chaetocnema pulicaria, corn flea; Sphenophorus

- 17 033842 maidis, corn weevil; Rhopalosiphum maidis; corn leaf aphid; Sipha flava, yellow sugarcane aphid; Blissus leucopterus leucopterus, North American wheat bug; Contarinia sorghicola, sorghum sorghum; Tetranychus cinnabarinus, a red spider mite; Tetranychus urticae, an ordinary spider mite; wheat: Pseudaletia unipunctata, meadow scoop; Spodoptera frugiperda, grass scoop; Elasmopalpus lignosellus, a small corn stalk grinder; Agrotis orthogonia, rectangular scoop; Elasmopalpus lignosellus, a small corn stalk grinder; Oulema melanopus, red-breasted drunkard; Hypera punctata, Clover leaf weevil; Diabrotica undecimpunctata howardi, southern corn root beetle; Russian wheat aphid; Schizaphis graminum, common cereal aphid; Macrosiphum avenae, large cereal aphid; Melanoplus femurrubrum, red-legged filly; Melanoplus differentialis, distinctive filly; Melanoplus sanguinipes, migratory filly; Mayetiola destructor, Hessian fly; Sitodiplosis mosellana, orange cereal gall midge; Meromyza americana, the larva of the American Meromiza; Hylemya coarctata, winter fly; Frankliniella fusca, tobacco thrips; Cephus cinctus, a bread sawfly; Aceria tulipae, an onion tick of tulips; Sunflower: Suleima helianthana, sunflower bud leaf; Homoeosoma electellum, sunflower moth; zygogramma exclamationis, sunflower exclamation scoop; Bothyrus gibbosus, carrot beetle; Neolasioptera murtfeldtiana, Galicia of sunflower seeds; Cotton: Heliothis virescens, cotton leaf; Helicoverpa zea, cotton scoop; Spodoptera exigua, small scoop; Pectinophora gossypiella, a pink cotton boxworm; Anthonomus grandis, cotton weevil; Aphis gossypii, cotton aphid; Pseudatomoscelis seriatus, cotton horsefly; Trialeurodes abutilonea, cotton whitefly; Lygus lineolaris, grass bug; Melanoplus femurrubrum, red-legged filly; Melanoplus differentialis, distinctive filly; Thrips tabaci, tobacco thrips; Franklinkiella fusca, tobacco thrips; Tetranychus cinnabarinus, a red spider mite; Tetranychus urticae, an ordinary spider mite; rice: Diatraea saccharalis, sugarcane grinder; Spodoptera frugiperda, grass scoop; Helicoverpa zea, corn scoop; rodent of the species Colaspis brunnea; Lissorhoptrus oryzophilus, rice water weevil; Sitophilus oryzae, rice weevil; Nephotettix nigropictus, rice circad; Blissus leucopterus leucopterus, North American wheat bug; Acrosternum hilare, a green bug bug; soybean: Pseudoplusia includens, soya moth; Anticarsia gemmatalis, a caterpillar of a velvet bean scoop; Plathypena scabra, a green clover pest; Ostrinia nubilalis, a European corn moth; Agrotis ipsilon, ipsilon scoop; Spodoptera exigua, small scoop; Heliothis virescens, cotton leaflet; Helicoverpa zea, corn scoop; Epilachna varivestis, Mexican bean kernel; Myzus persicae, green peach aphid; Empoasca fabae, potato cicadas; Acrosternum hilare, a green bug bug; Melanoplus femurrubrum, red-legged filly; Melanoplus differentialis, distinctive filly; Hylemya platura, sprout fly larva; Sericothrips variabilis, soybean thrips; Thrips tabaci, onion thrips; Tetranychus turkestani, a Turkestan spider mite; Tetranychus urticae, an ordinary spider mite; barley: Ostrinia nubilalis, a European corn moth; Agrotis ipsilon, ipsilon scoop; Schizaphis graminum, common cereal aphid; Blissus leucopterus leucopterus, North American wheat bug; Acrosternum hilare, a green bug bug; Euschistus servus, brown insect bug; Delia platura, sprout fly larva; Mayetiola destructor, Hessian fly; Petrobia latens, multinucleated petrobia; oilseed rape: Brevicoryne brassicae, cabbage aphid; Phyllotreta cruciferae, cruciferous flea Mamestra configurata, brass scoop; Plutella xylostella, cabbage scoop; Delia ssp., Larvae of spring cabbage fly.

Nematodes include parasitic nematodes, such as nodule, cyst-forming and damaging nematodes, including Heterodera spp., Meloidogyne spp. and Globodera spp .; in particular, representatives of cyst-forming nematodes, including, but not limited to, Heterodera glycines (soybean cyst-forming nematode); Heterodera schachtii (beet cyst-forming nematode); Heterodera avenae (cereal cyst-forming nematode); and Globodera rostochiensis and Globodera pailida (potato cyst-forming nematodes). Damaging nematodes include Pratylenchus spp.

Ways to increase plant yields

Methods for increasing plant yields are provided. The methods include providing a plant or plant cell that expresses a polynucleotide encoding the sequence of the pesticidal polypeptide disclosed herein, and growing the plant or seed obtained therefrom in a field infected (or susceptible to infection) by a pest with respect to which said polypeptide has pesticidal activity. In some embodiments, the polypeptide has pesticidal activity with respect to Lepidoptera, Coleoptera, Diptera, Hemoptera, or Nematode pest, and said field is infected with Lepidoptera, Semi-Rugged, Coleoptera, Diptera, or Nematode pest. As defined herein, plant yield refers to the quality and / or quantity of biomass produced by the plant. By biomass is meant any evaluated plant product. An increase in biomass production is any improvement in the measured yield of a plant product. Increasing plant yields has several commercial uses. For example, increasing the biomass of plant leaves can increase leaf yield

- 18,033,842 vegetables for human or animal consumption. Additionally, increasing leaf biomass can be used to increase the production of pharmaceutical or industrial plant products. The increase in yield may include any statistically significant increase, including, but not limited to, an increase of at least 1%, an increase of at least 3%, an increase of at least 5%, an increase of at least 10%, an increase of at least at least 20%, at least 30%, at least 50%, at least 70%, at least 100% or a more significant increase in yield compared to a plant in which a pesticidal protein sequence is not expressed. Using specific methods, plant yields are increased by increasing resistance to plant pests in which the pesticidal protein disclosed herein is expressed. The expression of the pesticidal protein reduces the ability of the pest to become infected or eaten.

Plants can also be treated with one or more chemical compositions containing one or more herbicides, insecticides or fungicides. Illustrative chemical compositions include: fruit / vegetable herbicides: atrazine, bromacil, diuron, glyphosate, linuron, metribuzin, simazine, trifluralin, fluazifop, glufosinat, Gowan halosulfuron, paraquat, propisamide, setoxydim, butafenulfamyl in galos; insecticides for fruits / vegetables: aldicarb, Bacillus thuriengiensis, carbaryl, carbofuran, chlorpyrifos, cypermethrin, deltamethrin, abamectin, cyfluthrin / beta-cyfluthrin, esfenvalerate, lambda-cyhalothrin, atsekvinotsil, bifenazate, methoxyfenozide, novaluron, chromafenozide, thiacloprid, dinotefuran, fluatsripirim , spirodiclofen, gamma cygalotrin, spiromesifen, spinosad, rinaxipyr, cyazipyr, triflumuron, spiro tetramate, imidacloprid, flubendiamide, thiodicarb, metaflumizone, sulfoxaflor, tsiflumetofen, cyanopyrafenet, tylotamidin, tylotani, tylotani flonicamide, methiocarb, emamectin benzoate, indoxacarb, fenamifos, pyriproxifen, fenbutatin oxide; fungicides for fruits / vegetables: amethoctradine, azoxystrobin, bentiavalicarb, boscalide, captan, carbendazim, chlorothalonil, copper, cyazofamide, tsiflufenamid, cymoxanil, ciproconazole, cyprodinil, diphenoconazole, dimethomorphidopyldamidone fidiamido phyldiamine didione, dimethanfludiamidopyldione, dithiomendiamidopyldione, dithiomeldiamidopyldamidone, dimethyldiamine, dithiomeldiamidopyldione, fluoxastrobin, fluxapyroxad, folpet, fosetil, iprodion, iprovalicarb, isopyrazam, kresoxim-methyl, mancozeb, mandipropamide, metalaxyl / mefenoxam, metiram, metrafenone, miklobutanil, penconazole, pentiopyrobram, propoxypropamide, pic konazol, propineb, prokvinazid, prothioconazole, pyraclostrobin, pyrimethanil, quinoxyfen, spiroxamine, sulfur, tebuconazole, thiophanate-methyl, trifloxystrobin; cereal herbicides: 2,4-D, amidosulfuron, bromoxynil, carfentrazone ethyl, chlorotoluron, chlorosulfuron, clodinafop-, clopyralide, dicamba, diclofop-methyl, diflufenican, fenoxaprop, flurasulam, flucarburofurfonfenfurfonfurfonfurfonfonet, flucarburofurfonfenfon, flucarburofurfonfenfen, , glyphosate, iodosulfuron, ioxynil, isoproturon, MCPA, mesosulfuron, metsulfuron, pendimethalin, pinoxaden, propoxycarbazone, prosulfocarb, pyroxulam, sulfosulfuron, tifensulfuron, tralkoxydim, triasulfuron, trien; fungitsidydlya cereals: azoxystrobin, biksafen, boscalid, carbendazim, chlorothalonil, cyflufenamid, cyproconazole, cyprodinil, dimoxystrobin, epoxiconazole, fenpropidin, fenpropimorph, fluopiram, fluoxastrobin, fluquinconazole, fluksapiroksad, izopirazam, kresoxim-methyl, metconazole, metrafenone, penthiopyrad, picoxystrobin, prochloraz , propiconazole, proquinazide, prothioconazole, pyraclostrobin, quinoxifene, spiroxamine, tebuconazole, thiofanate methyl, trifloxystrobin; cereal insecticides: _dimethoate, lambda-cygalothrin, deltamethrin, alpha-cypermethrin, β-tsiflutrin, bifentrin, imidacloprid, clothianidin, tiameto xam, thiacloprid, acetamipride, dinetofuran, chlorpyrififos, methyrimocir herbicides for maize: _atrazine, alachlor, bromoxynil, acetochlor, dicamba, clopyralid, (B-) dimethenamide, glufosinate, glyphosate, isoxaflutol, (B-) metolachlor, mesotrione, nicosulfuron, primisulfuronramonum, primisulfuronramonum, topical sulfulfuron rimon, topical sulfulfuron rimon, topical sulfulfuron rimon, topical sulfuron rimon saflufenacil, thiencarbazone, flufenacet, pyroxasulfone; insecticides for maize: _karbofuran, chlorpyrifos, bifenthrin, fipronil, imidacloprid, lambda-cyhalothrin, tefluthrin, terbufos, thiamethoxam, clothianidin, spiromesifen, flubendiamide, triflumuron, rinaksipir, deltamethrin, thiodicarb, β-cyfluthrin, cypermethrin, bifenthrin, lufenuron, tebupirimfos, etiprol, cyazipyr, thiacloprid, acetamiprid, dinetofuran, avermectin; fungicides for maize: _azoxystrobin, bixafen, boscalide, ciproconazole, dimoxystrobin, epoxiconazole, phenitropan, fluopyram, fluoxastrobin, fluxapiroxade, isopyrazam, metconazole, pentiopyrad, picoxystrobolazole, propoxynobrazole, propoxyprolazole, propoxyprobolone, propoxyprobolone herbicides for rice: butachlor, propanil, azimsulfuron, bensulfuron, cyhalofop, daimuron, fentrazamid, imazosulfuron, mefenacet, oxaziclomefone, pyrazosulfuron, pyributicarb, quinclorac, thiobencarb, indanofan, flufenacet, fentrazamid, halosulfuron, oxaziclomefone, benzobitsiklon, pyriftalid, penoxsulam, bispyribac, oxadiargil, ethoxysulfuron, pretilachlor, mesotrione, tefuryltrione, oxadiazone, fenoxaprop, pyrimisulfan; rice insecticides: _diazinon, phenobucarb, benfuracarb, buprofesin, dinotefuran, fipronil, imidacloprid, isoprocarb, thiacloprid, chromafenoside, clothianidine, etiprol, flubendiamide, rinaxipyr, benzidetamide tampertamide tampertamide tampertamide tampertamide tampertamide tampertamide, tamethamethamide, trimethamide tampertamide, trimethamide, trimethamide chlorpyrifos, etofenprox,

- 19 033842 carbofuran, benfuracarb, sulfoxaflor; rice fungicides: _azoxystrobin, carbendazim, carpropamide, diclocimet, diphenoconazole, edifenfos, ferimzon, gentamicin, hexaconazole, gimexazole, iprobenfos (IPB), isoprothiolan, propanofinone, probotericene, pomegrobenicin azinobromazoline, manglozenesin, , tebuconazole, thiofanate methyl, thiadinyl, tricyclazole, trifloxystrobin, validamycin; cotton herbicides: diuron, fluometuron, MSMA, oxyfluorfen, prometrin, trifluralin, carfentrazone, glucodim, fluazifop-butyl, glyphosate, norflurazone, pendimethalin, pyrithiobac-sodium, trifloxisulfuron, teflazinum, teralazin; cotton insecticides: _acecefate, aldicarb, chlorpyrifos, cypermethrin, deltamethrin, abamectin, acetamiprid, emamectin benzoate, imidacloprid, indoxacarb, lambdacigalotrin, spinosad, thiodicarbide, pyridimeldyridenfiridimeridifridedrydimeldiurifide-di-frimedinifridedifrideldifrideldifridedifrideldifrideldifrideldifrideldimeridinifridedimeridin , spiro tetramate, clothianidin, thiamethoxam, thiacloprid, dinetofuran, flubendiamide, cyazipyr, spinosad, spinotoram, gamma-cygalotrin, 4 - [[(6chloropyridin-3-yl) methyl] (2,2-difluoroethyl) amino] furan-2 (5 ) -one, thiocarb, avermectin, flonicamide, pyridinyl, spir mesifen, sulfoksaflor; cotton fungicides: azoxystrobin, bixafen, boscalide, carbendazim, chlorothalonil, copper, cyproconazole, diphenoconazole, dimoxystrobin, epoxiconazole, phenamidone, fluazinam, fluopyramopyroxinoprobin pirobibrobinoprodibiroprobinoprodibiropinoprodibiropinoprodibiropinoprodibiropinopromen propineb, prothioconazole, pyraclostrobin, quintozene, tebuconazole, tetraconazole, thiofanate methyl, trifloxystrobin; soybean herbicides: alachlor, bentazone, trifluralin, chlorimuron-ethyl, chloransulam-methyl, fenoxaprop, fomesafen, fluazifop, glyphosate, imazamox, imazakvin, imazetapir, ^ -) metolachlor, metribuzin, pendimethymaline, tepral; soy insecticides: _lambda-cygalotrin, methomil, imidacloprid, clotianidin, thiamethoxam, thiacloprid, acetamipride, dinetofuran, flubendiamide, rinaxipyr, cyazipyr, spinosad, spinetoram, emamectin-benzoate, cyprimytrind, gamma-typrin, pyramyrrind, gamma-typrin, pyramyrgind, gamma-typrilipyrmind, gamma-typrilipyrmind, gamma-pyrrolitha, gamma typrin, pyramyrind , 4 - [[(6-chloropyridin-3-yl) methyl] (2,2-difluoroethyl) amino] furan-2 (5H) -one, spiro tetramate, spinodiclofen, triflumuron, flonicamide, thiodicarb, beta-cyfluthrin; soybean fungicides: _azoxystrobin, bixafen, boscalide, carbendazim, chlorothalonil, copper, cyproconazole, diphenoconazole, dimoxystrobin, epoxiconazole, fluazinum, fluopyram, fluoxastrobin, flutriofoloxinobroziobrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrozopyrodin pentiopyrad, picoxystrobin, propiconazole, propineb, prothioconazole, pyraclostrobin, tebuconazole, tetraconazole, thiofanate methyl, trifloxystrobin; sugar beet herbicides: chloridazone, desmedifam, etofumezate, fenmedifam, triallate, clopyralide, fluazifop, lenacyl, metamitron, quinmerac, cycloxydim, triflusulfuron, teraloksidim, chizalofop; sugar beet insecticides: _imidacloprid, clotianidin, thiamethoxam, thiacloprid, acetamiprid, dinetofuran, deltamethrin, β-cyclifutrin, gamma / lambda-cyhalothrin, 4 - [[(6-chloropyridin-3-yl) methyl] (2,2-aminorithi ] furan2 (5H) -one, teflutrin, rinaxipyr, tsiaksipir, fipronil, carbofuran; canola herbicides: clopyralid, diclofop, fluazifop, glufosinate, glyphosate, metazachlor, trifluralin etametsulfuron, quinmerac, chizalofop, Clethodim, teraloksidim; fungicides for canola: _azoxystrobin, bixafen, boscalide, carbendazim, ciproconazole, diphenoconazole, dimoxystrobin, epoxiconazole, fluazinam, fluopyram, fluoxastrobin, flusilazoloprozoprazinoprozoprazinoprozoprozomoprozomoprozomoprozomoprozomoprozomoprozomoprozomoprozomoprozomoprozomoprozomoprozomoprozomoprozomoprozomprazod prothioconazole, pyraclostrobin, tebuconazole, thiofanate methyl, trifloxystrobin, vinclozolin; insecticides for canola: _carbofuran, thiacloprid, deltamethrin, imidacloprid, clotianidin, thiamethoxam, acetamiprid, dinetofuran, β-cyfluthrin, gamma and lambda-cygalotrin, tau-fluvaleriate, ethiprol, spinosadinamide di-azideamide, spinetamide, spinetamide, spinetamide [(6-chloropyridin-3-yl) methyl] (2,2-difluoroethyl) amino] furan-2 (5H) -one.

The following examples are provided for purposes of illustration and not for purposes of limitation.

Experimental examples

Example 1. The discovery of new genes that determine pesticidal activity in Bacillus thuringiensis.

New pesticidal genes were identified from the bacterial strains ATX47307 and ATX56002 using the following steps.

Getting all the DNA from the strain. Total DNA contains both genomic DNA and extrachromosomal DNA. Extrachromosomal DNA contains a mixture of some or all of the following: plasmids of various sizes; phage chromosomes; other uncharacterized extrachromosomal molecules.

DNA sequencing. Total DNA is sequenced using next generation sequencing methods.

Identification of putative toxin genes through homology analyzes and / or other computational analyzes

Obtaining the final specified sequence of the gene of interest by one of several PCR or cloning strategies (e.g., TAIL-PCR), if necessary.

- 20 033842

Table 1. The new gene identified in the strain ATX47307

Gene name Molecular weight (kDa) Closest homologue Nucleotide SEQ ID NO Amino Acid SEQ ID NO Axmi477 132 75% SGU9VA1 1 5 Axmi477.2 6 Axmi477.3 7 Ahsh1477 (truncated) 60% Cry9Bal (truncated) 8 Ahsh1477.2 (truncated) 9 Ahsh1477.3 (truncated) 10

Axmi477.2 and Axmi477.3 are proteins encoded from the downstream portion of the start of translation relative to Axmi477.

Table 2. The new gene identified in the strain ATX47307

Gene name Molecular Weight (kDa) Closest homologue Nucleotide SEQ ID NO Amino Acid SEQ ID NO Axmi482 37 94% Axmi486; 51% Mtx3 2 eleven Axmi482.2 12 Axmi482.3 thirteen Axmi482.4 14

Axmi482.2, Axmi482.3, and Axmi482.4 are proteins encoded from the translation start portion of the relative start of Axmi482.

Table 3. New gene identified in strain ATX65002

Gene name Molecular Weight (kDa) Closest homologue Nucleotide SEQ ID NO Amino Acid SEQ ID NO Axmi486 38 94% Axmi482; 49% Mtx3 3 fifteen Axmi486.2 16 Axmi486.3 17 Axmi486.4 18 Axmi486.5 19

Axmi486.2, Axmi486.3, Axmi486.4, and Axmi486.5 are proteins encoded from the translation start portion that is lower than the Axmi486.

Table 4. New gene identified in strain ATX65002

Gene name Molecular Weight (kDa) Closest homologue Nucleotide SEQ ID NO Amino Acid SEQ ID NO > Axmi525 > Axmi525.2 > Axmi525.3 > Axmi525.4 > Axmi525.5 > Axmi525.6 > Axmi525.7 38 97% Axmi486; 51% Mtx3 4 20 21 22 23 24 25 26

Axmi525.2, Axmi525.3, Axmi525.4, Axmi525.5, Axmi525.6, and Axmi525.7 are proteins encoded from the translation start portion that is lower than the Axmi525.

Example 2. Analyzes of pesticidal activity.

The nucleotide sequences of the present invention can be examined with respect to their ability to produce pesticidal proteins. The ability of a pesticidal protein to act on a pest as a pesticide is often analyzed using a number of methods. One method well known in the art is to perform feeding analysis. In this assay, the pest is exposed to a sample containing either the compounds to be tested or control samples. Often it is carried out when placing the material that needs to be investigated, or a suitable solution of such material on a material that the pest will absorb, such as an artificial nutrient medium. The material to be examined may be composed of liquid, solid, or suspension. The material to be examined can be placed on the surface and then allowed to dry. Alternatively, the material to be investigated can be mixed with the molten artificial nutrient medium and then distributed in the analysis chamber. The analysis chamber may be, for example, a cup, plate or well of a microtiter plate.

Assays with sucking pests (such as aphids) may include separating the test material from the insect by a partition, ideally a section that the sucking insect can pierce with parts of the sucking mouth to absorb the test material. Often, test material is mixed with an eating stimulant, such as sucrose, to facilitate uptake of the test compound.

Other types of assays may include microinjection of the test material into the pest's oral cavity or intestines, as well as the development of transgenic plants, followed by testing the pest's ability to feed on the transgenic plant. Plant research may include isolate

- 21 033842 parts of plants that are usually consumed, for example, in small chambers attached to a leaf, or isolation of whole plants in chambers that contain insects.

Other methods and approaches for assessing pests are known in the art and can be found, for example, from Robertson and Preisler, eds. (1992) Pesticide bioassays with arthropods CRC, Boca Raton, FL. Alternatively, analyzes are usually described in the Arthropod Management Tests and Journal of Economic Entomology or discussed with members of the Entomological Society of America (ESA).

In some embodiments, the DNA regions encoding the toxin region in the pesticidal proteins disclosed herein are cloned into the E. coli pMAL-C4x expression vector for the malE gene encoding the maltose binding protein (MBP). These intra-frame fusions result in the expression of MBP-Axmi fusion proteins in E. coli.

For expression in E. coli, BL21 * DE3 is transformed with individual plasmids. Separate colonies were inoculated into Luria-Bertani medium supplemented with carbenicillin and glucose and grown overnight at 37 ° C. The next day, fresh medium is inoculated with 1% of the daily culture and grown at 37 ° C until the logarithmic growth phase. Then, cultures were induced with 0.3 mM IPTG overnight at 20 ° C. Each cell pellet was suspended in 20 mM Tris-Cl buffer, pH 7.4 + 200 mM NaCl + 1 mM DTT + protease inhibitors and destroyed by ultrasound. To confirm expression of the fusion proteins, an analysis using SDS-PAGE can be used.

All cell-free extracts are then run through an amylose column attached to a liquid chromatography rapid protein chromatograph (FPLC) for the affinity purification of MBP-axmi fusion proteins. Bound fusion proteins are eluted from the resin using a 10 mM maltose solution. The purified fusion proteins are then cleaved with either factor Xa or trypsin to remove the amino terminal MBP tag from the Axmi protein. Protein cleavage and solubility can be determined using SDS-PAGE.

Example 3. Expression and purification.

Truncated versions of Axmi477 (which is set forth herein as SEQ ID NO: 8), Axmi482 (which is set forth herein as SEQ ID NO: 13), Axmi486 (which is set forth as SEQ ID NO: 16), and Axmi525 (which set forth herein as SEQ ID NO: 26) were expressed and analyzed for biological activity. Genes were amplified by PCR from their respective strains using HERCULASE® II fusion DNA polymerase with primers with integrated AscI linkers at the 3'-end. The amplified PCR product was digested with AscI and ligated into the pMalC4X vector. Cloning was confirmed by sequencing and transformed into competent Bl21 cells. Each individual colony was inoculated into Luria-Bertani medium and grown at 37 ° C to the log phase, and 0.5 mM IPTG was induced at 20 ° C for 18 hours. The purified protein was digested with factor Xa at a ratio of 1:50 at room temperature overnight. The purified protein was analyzed for biological activity against selected pests according to a standard protocol. The results are shown in table. 5-8.

Table 5. Mortality and stunting rates for Axmi477

Pest group Growth retardation rate Mortality, in percent Plutella xylostella (DBM) 4 100 Anticarsia gemmatalis (VBC) 4 100 Diatraea grandiosella (SWCB) 3,5 25 Diatraea saccharalis (SCB) 3,5 0 Heliothis virescens (Hv) 4 75 Heliocoverpa zea (Hz) 2 25 Ostrinia nublialis (ECB) 3 25 Spodoptera frugiperda (FAW) 1 0 Spodoptera exigua (BAW) 3 0 Agrotis ipsilon (BCW) 4 0 Pseudoplusia includens (SBL) 4 100

Table 6. Mortality and stunting rates for Axmi482

Pest group Growth retardation rate Mortality, in percent Plutella xylostella (DBM) 4 100 Anticarsia gemmatalis (VBC) 4 75 Diatraea grandiosella (SWCB) 4 fifty Diatraea saccharalis (SCB) 3 0 Heliothis virescens (Hv) 1 0 Heliocoverpa zea (Hz) 4 0 Ostrinia nublialis (ECB) 3 1 Spodoptera frugiperda (FAW) 3 0

- 22 033842

Table 7. Mortality and stunting rates for Axmi486

Pest group Growth retardation rate Mortality, in percent Plutella xylostella (DBM) 4 100 Anticarsia gemmatalis (VBC) 4 37 Diatraea grandiosella (SWCB) 3,5 fifty Diatraea saccharalis (SCB) 2,5 25 Heliothis virescens (Hv) 3,5 0 Heliocoverpa zea (Hz) 3 0 Pseudoplusia includens (SBL) 1 0

Table 8. Mortality and stunting rates for Axmi525

Pest group Growth retardation rate Mortality, in percent Spodoptera frugiperda (FAW) 1 0% Heliothis virescens (Hv) 1,5 0% Helicoverpa zea (Hz) 3 0% Anticarsia gemmatalis (VBC) 4 42% Spodoptera eridania (SAW) 3.2 70% Plutella xylostella (DBM) 4 100% Diatraea grandiosella (SWCB) 4 83% Diatraea crambidoides (SCB) 4 66%

Growth retardation rate:

- No activity.

- Non-uniform growth retardation.

- Slight growth retardation.

- Strong growth retardation.

- A sharp growth retardation.

Example 4. Vectoring of Genes for Plant Expression

The coding regions of the present invention are combined with the corresponding sequences of promoters and terminators for expression in plants. Such sequences are well known in the art and may include the rice actin promoter or maize ubiquitin promoter for expression in monocotyledonous plants, the Arabidopsis UBQ3 promoter or CaMV 35S promoter for expression in dicotyledonous plants, and nos or PinII terminators. Methods for obtaining and confirming promoter - gene - terminator constructs are also well known in the art.

In one aspect of the present invention, synthetic DNA sequences are designed and created. These synthetic sequences have a modified nucleotide sequence compared to the original sequence, but encode proteins that are essentially identical to the original sequence.

In another aspect of the present invention, modified versions of synthetic genes are designed such that the resulting peptide targets a plant organelle, such as an endoplasmic reticulum or apoplast. Sequences of peptides are known from the prior art that are known to result in the targeting of fusion proteins to plant organelles. For example, it is known in the art that the N-terminal portion of a protein encoded by the white lupine acid phosphatase gene Lupinus albus (GENBANK® ID GI: 14276838, Miller et al. (2001) Plant Physiology 127: 594-606) leads to heterologous targeting proteins on the endoplasmic reticulum. If the resulting fusion protein also contains a sequence at the C-terminus for retention in the endoplasmic reticulum containing lysine-aspartic acid-glutamic acid-leucine from the N-terminus of the peptide (i.e., KDEL motif, SEQ ID NO: 27), then the fusion protein will target the endoplasmic reticulum. If there is no sequence in the fusion protein at the C-terminus that targets the endoplasmic reticulum, the protein will target the endoplasmic reticulum, but will eventually be bound in the apoplast.

Thus, this gene encodes a fusion protein that contains at the N-terminus thirty-one amino acids from the white lupine acid phosphatase gene Lupinus albus (GENBANK® ID GI: 14276838, Miller et al., 2001, see above), fused to N- the end of the amino acid sequence of the present invention, as well as the KDEL sequence (SEQ ID NO: 27) at the C-terminus. Thus, it is assumed that the resulting protein targets the endoplasmic reticulum of the plant when expressed in the plant cell.

The plant expression cassettes described above are combined with a plant selectable marker to facilitate selection of transformed cells and tissues, and ligated into vectors for plant transformation. These may include binary vectors for Agrobacterium mediated transformation or simple plasmid vectors for transformation using aerosol or biolistic transformation.

Example 5. Transformation of maize cells with the pesticidal protein genes described herein.

- 23,033,842

Maize ears are best harvested 8-12 days after pollination. The embryos are isolated from the cobs and embryos 0.8-1.5 mm in size are preferably used during transformation. Embryos are planted upside down in a suitable incubation medium, such as DN62A5S medium (3.98 g / l N6 salts; 1 ml / l (1000x stock solution), vitamins N6; 800 mg / l L-asparagine; 100 mg / l myoinositis ; 1.4 g / l of L-proline; 100 mg / l of casamic acids; 50 g / l of sucrose; 1 ml / l (stock solution with a concentration of 1 mg / ml) 2,4-D). However, media and salts other than DN62A5S and known in the art are suitable. The embryos are incubated overnight at 25 ° C in the dark. However, incubation of the embryos during the night as such is not necessary.

The resulting explants are transferred to mesh cells (30-40 per dish), transferred to the osmotic medium for about 30-45 minutes, then transferred to the dish beam (see, for example, PCT Publication No. WO / 0138514 and US Patent No. 5240842).

DNA constructs designed for expression of the genes of the present invention in plant cells are obtained by acceleration into plant tissue using an aerosol beam accelerator, using conditions that are essentially described in PCT publication No. WO / 0138514. After exposure to the beam, the embryos are incubated for about 30 minutes in an osmotic medium and placed in an incubation medium overnight at 25 ° C in the dark. To avoid excessive damage to the explants exposed to the beam, they are incubated for at least 24 hours before being transferred to the recovery medium. The embryos are then distributed in the medium for a recovery period of approximately 5 days at 25 ° C. in the dark, then transferred to a selective medium. Explants are incubated in selective medium for a period of up to eight weeks, depending on the nature and characteristics of the particular selection method used. After the selection period, the resulting callus is transferred to the embryo maturation medium and cultured until the formation of mature somatic embryos is observed. The resulting mature somatic embryos are then placed under low light conditions and the regeneration process is initiated using methods known in the art. The resulting shoots are allowed to take root on the rooting medium, and the resulting plants are transferred to seedling pots and propagated as transgenic plants.

Materials

Medium DN62A5S

Components Per liter A source A mixture of basic salts N6 Chu (product number C 416) 3.98 g / l Phytotechnology labs Chu Vitamin N6 Solution (Product No. C 149) 1 ml / l (1000x stock solution) Phytotechnology labs L-asparagine 800 mg / l Phytotechnology labs myoinositis 100 mg / l Sigma L-proline 1.4 g / l Phytotechnology labs casamic acids 100 mg / l Fisher scientific sucrose 50 g / l Phytotechnology labs 2,4-D (product number D- 7299) 1 ml / l (stock solution with a concentration of 1 mg / ml) Sigma

The pH of the solution was adjusted to pH 5.8 with 1N KOH / 1N KCl, Gelrite (Sigma) was added at a concentration of 3 g / L and the medium was autoclaved. After cooling to 50 ° C., 2 ml / l of a stock solution of silver nitrate with a concentration of 5 mg / ml is added (Phytotechnology Labs).

Example 6. Gene transformation according to the present invention of plant cells by Agrobacterium mediated transformation.

Ears are best harvested 8-12 days after pollination. The embryos are isolated from the cobs and embryos 0.8-1.5 mm in size are preferably used during transformation. The embryos are planted upside down in a suitable incubation medium and incubated overnight at 25 ° C in the dark. However, incubation of the embryos during the night as such is not necessary. The embryos are brought into contact with an Agrobacterium strain containing vectors suitable for Ti plasmid-mediated transfer for about 5-10 minutes and then placed in co-culture medium for about 3 days (25 ° C. in the dark). After cocultivation, the explants are transferred to the medium for a recovery period of approximately five days (at 25 ° C in the dark). Explants are incubated in selective medium for a period of eight weeks, depending on the nature and characteristics of the particular selection method used. After a selection period, the resulting callus is transferred to the embryo maturing medium and cultured until the formation of mature somatic embryos is observed. The resulting mature somatic embryos are then placed under low light conditions and initiate the regeneration process, as is known in the art.

All publications and patent applications mentioned in this description indicate the qualifications of specialists in the art to which the present invention belongs. All publications and patent applications are incorporated herein by reference to the same extent as if

- 24,033,842, it was understood that each individual publication or patent application is specifically and separately incorporated by reference.

Although the foregoing invention has been described in sufficient detail for the purpose of illustration, and also as an example for clarity of understanding, it is obvious that in practice, certain changes and modifications can be made within the scope of the attached claims.

Claims (23)

1. A recombinant nucleic acid molecule that contains a nucleotide sequence encoding a protein having pesticidal activity against a lepidopteran pest, selected from the group consisting of: a) the nucleotide sequence of SEQ ID NO: 1; b) a nucleotide sequence that encodes a polypeptide containing an amino acid sequence selected from SEQ ID NO: 5-10; c) a nucleotide sequence that encodes a polypeptide containing an amino acid sequence that is at least 95% identical to the sequence selected from SEQ ID NO: 5-10. 2. The recombinant nucleic acid molecule according to claim 1, which is a synthetic sequence that was designed for expression in a plant. 3. The recombinant nucleic acid molecule according to claim 1, operably linked to a promoter capable of controlling the expression of said protein in a plant cell. 4. A vector containing the recombinant nucleic acid molecule according to claim 1. 5. A host cell that contains the recombinant nucleic acid according to claim 1. 6. The host cell according to claim 5, which is a bacterial cell. 7. The host cell according to claim 5, which is a plant cell. 8. A transgenic plant that contains a host cell according to claim 7, which is resistant to the pest of the order Lepidoptera. 9. The transgenic plant of claim 8, selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, cruciferous, pepper, potato, cotton, rice, soy, sugar beet, sugarcane, tobacco, barley and oilseed rapeseed. 10. Transgenic seed containing a nucleic acid molecule according to claim 1. 11. Recombinant polypeptide having pesticidal activity against lepidopteran pest, selected from the group consisting of: a) a polypeptide containing an amino acid sequence selected from SEQ ID NO: 510, and b) a polypeptide containing an amino acid sequence that is at least 95% identical to the sequence selected from SEQ ID NO: 5-10. 12. The composition containing the polypeptide according to claim 11, having activity against the pest of the order Lepidoptera. 13. The composition according to p. 12 in the form of a powder, dust, pellets, granules, aerosol, emulsion and solution, including colloid. 14. The composition according to item 12, obtained by drying, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation or concentration of a bacterial cell culture. 15. The composition according to item 12, containing a mass fraction of from about 1% to about 99% of the specified polypeptide. 16. A method of controlling a population of Lepidoptera pest, comprising bringing the specified population into contact with a pesticidally effective amount of the polypeptide according to claim 11. 17. A method of destroying a lepidopteran pest, comprising bringing said pest into contact with a pesticidally effective amount of a polypeptide according to claim 11. 18. A method of producing a polypeptide with pesticidal activity, comprising culturing a host cell according to claim 5 under conditions providing expression of a nucleic acid molecule encoding the specified polypeptide. 19. A plant with a nucleic acid molecule stably integrated in its genome containing a nucleotide sequence that encodes a protein having pesticidal activity against a lepidopteran pest selected from the group consisting of: a) the nucleotide sequence of SEQ ID NO: 1; b) a nucleotide sequence that encodes a polypeptide containing an amino acid sequence selected from SEQ ID NO: 5-10, and c) a nucleotide sequence that encodes a polypeptide containing an amino acid sequence that is at least 95% identical to the sequence selected from SEQ ID NO: 5-10. 20. A plant cell with a nucleic acid molecule stably integrated in its genome containing - 25 033842 containing the nucleotide sequence that encodes a protein having pesticidal activity against lepidopteran pest selected from the group consisting of: a) the nucleotide sequence of SEQ ID NO: 1; b) a nucleotide sequence that encodes a polypeptide containing an amino acid sequence selected from SEQ ID NO: 5-10, and c) a nucleotide sequence that encodes a polypeptide containing an amino acid sequence that is at least 95% identical to the sequence selected from SEQ ID NO: 5-10. 21. A method of protecting a plant from a pest, comprising expressing a nucleic acid molecule in a plant or its cell that encodes a polypeptide having pesticidal activity against a lepidopteran pest having a nucleotide sequence selected from the group consisting of: a) the nucleotide sequence of SEQ ID NO: 1; b) a nucleotide sequence that encodes a polypeptide containing an amino acid sequence selected from SEQ ID NO: 5-10, and c) a nucleotide sequence that encodes a polypeptide containing an amino acid sequence that is at least 95% identical to the sequence selected from SEQ ID NO: 5-10. 22. The method according to p. 21, characterized in that said plant produces a polypeptide having pesticidal activity against Lepidoptera, Hemoptera, Coleoptera, and nematodes or diptera. 23. A method of increasing the yield of a plant, including growing in the field of a plant or its seed with a nucleic acid molecule stably integrated in its genome containing a nucleotide sequence that encodes a protein having pesticidal activity selected from the group consisting of: a) the nucleotide sequence of SEQ ID NO: 1; b) a nucleotide sequence that encodes a polypeptide containing an amino acid sequence selected from SEQ ID NO: 5-10, and c) a nucleotide sequence that encodes a polypeptide containing an amino acid sequence that is at least 95% identical to the sequence selected from SEQ ID NO: 5-10, wherein said field is infected by a pest with respect to which said polypeptide exhibits pesticidal activity. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2